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Bio<br />

05<br />

Next generation<br />

PCR<br />

New RainDrop System surpasses all<br />

existing digital PCR technologies<br />

Achieve accurate<br />

PCR results<br />

Obtaining optimal and reproducible<br />

PCR conditions<br />

Monitoring Tumour<br />

Metastases &<br />

Osteolytic Lesions<br />

With Bioluminescence & MicroCT Imaging


Offices & Service Centers<br />

Australia<br />

Head Office:<br />

Street Address: 5 Caribbean Dr, Scoresby,<br />

Victoria 3179<br />

Postal Address: PO Box 9092,<br />

Scoresby, Vic 3179<br />

Telephone Number: 1300-735-292<br />

AU Service Centre Locations:<br />

Melbourne: Scoresby<br />

Sydney: North Ryde<br />

Brisbane: Richlands<br />

South Australia: Thebarton<br />

Perth: Malaga<br />

New Zealand<br />

Head Office:<br />

Street Address: 244 Bush Road,<br />

Albany, North Shore City, 0632,<br />

Postal Address: Private Bag 102922,<br />

North Shore, North Shore City, 0745<br />

Telephone Number: 0800-933-966<br />

Welcome<br />

This publication marks our fifth edition of the Bio-Innovation<br />

series, a magazine dedicated to discussing new and emerging<br />

trends relevant to the field of life science for our Australian and<br />

New Zealand customers.<br />

This issue is filled with new developments in the field of PCR<br />

(Polymerase Chain Reaction) - a technique established over 30<br />

years ago and commonplace in nearly all research laboratories<br />

today. We focus on several exciting new developments that<br />

include the ability to directly amplify DNA from tissue samples<br />

right through to conducting massively parallel screens of up to<br />

20,000 unique primer pairs in a standard reaction volume and<br />

reporting out the results with absolute quantification. Welcome to<br />

the dawn of direct and digital PCR…<br />

NZ Service Centre Locations:<br />

Auckland: Albany<br />

Christchurch: Wigram<br />

Palmerston North<br />

Wellington: Lower Hutt<br />

Finally, following the trend of transitioning research from bench<br />

to bedside, we discuss advances in exploring for diagnostic and<br />

theragnostic markers in patients with Alzheimer’s disease and<br />

the ability to longitudinally monitor tumour metastases using<br />

bioluminensense and microCT imaging. Enjoy!<br />

Editor<br />

Mika Mitropoulos: mika.mitropoulos@thermofisher.com<br />

Art & Design<br />

Andrew Dennis: andrew.dennis@thermofisher.com<br />

Raina Macpherson: raina.macpherson@thermofisher.com<br />

Reno Leuc: reno.leuc@thermofisher.com<br />

Russell Twidle: russell.twidle@thermofisher.com<br />

Cover Image: RNA transcription. Computer artwork showing transcription of<br />

RNA (ribonucleic acid) within a human cell. Transcription is the process of creating<br />

a complementary RNA copy of a sequence of DNA (deoxyribonucleic acid,<br />

blue). During transcription the DNA sequence is 'unzipped' (centre) and 'read' by<br />

an RNA polymerase enzyme (purple), which then produces the complementary<br />

strand of RNA (green) by combining the appropriate nucleotides.<br />

Harald Ottenhof (PhD)<br />

Business Development Manager<br />

Life Science<br />

<strong>Thermo</strong> <strong>Fisher</strong> Scientific


Contents<br />

Next generation PCR 04<br />

What is digital PCR? 05<br />

Achieve accurate PCR results 08<br />

Direct PCR: genotyping transgenic mice 10<br />

Monitoring Tumour Metastases 12<br />

Prevent sample loss during cryostorage 15<br />

A targeted Aβ proteomic platform for Alzheimer’s' disease 16<br />

Solution for RT-qPCR 24<br />

Reverse transcription enzymes 26<br />

Pioneering in PCR Technology 28<br />

DNA quantification in micro-litre volumes 30<br />

High content gene expression analysis 32<br />

Separation of influenza virus 34<br />

High throughput RNA isolation 36<br />

Q&A 39


Next generation PCR<br />

RainDance launches the new RainDrop<br />

New RainDrop System surpasses all existing digital PCR technologies<br />

and establishes new performance standards in cancer research<br />

RainDance Technologies, Inc., the Digital Biology company,<br />

has introduced its new RainDrop Digital PCR System,<br />

which establishes new performance standards in sensitivity,<br />

multiplexing and absolute quantitation in PCR analysis.<br />

Capable of generating more than a billion reactions in a single<br />

day, the RainDrop System transforms the performance of<br />

molecular assays by enabling digital answers across a number<br />

of important applications including low-frequency tumour allele<br />

detection, gene expression, copy number variation, and SNP<br />

measurement.<br />

Built on RainDance’s patented and proven RainStorm<br />

picodroplet technology, the RainDrop System generates up to<br />

10 million picoliter-sized droplets per lane. Since each droplet<br />

encapsulates a single molecule, researchers can quickly<br />

determine the absolute number of droplets containing specific<br />

target DNA and compare that to the number of droplets<br />

with background wild-type DNA. The RainDrop System<br />

also shifts the current digital PCR (dPCR) paradigm from a<br />

single-colour-per-marker approach to a two colour and varying<br />

probe intensity method that is capable of multiplexing up to 10<br />

markers.<br />

In a recent Lab on a Chip paper, scientists from Université<br />

de Strasbourg and Université Paris Descartes, used the<br />

RainDance dPCR technology to detect a single mutated<br />

copy of KRAS in a background of 200,000 wild-type copies.<br />

By processing reactions in millions of picoliter droplets, the<br />

platform improved sensitivity by two orders of magnitude<br />

compared to existing technologies.<br />

“With RainDance digital PCR, we were able to achieve absolute<br />

quantification of mutated and tumour-circulating DNA and<br />

improve the detection of a circulating tumour by comparing<br />

its proportion to non-tumour DNA,” added Professor Pierre<br />

Laurent-Puig, M.D., Ph.D. of the Université Paris Descartes.<br />

“Absolute quantification is critical, especially in research that<br />

lays the groundwork for future clinical applications, because<br />

it allows you to generate meaningful thresholds that will be<br />

required for prognostic and diagnostic tools.”<br />

“Two years ago, RainDance embarked on an ambitious goal<br />

when it set out to develop a digital PCR technology platform<br />

that would fundamentally transform cancer research, and<br />

open the door to new ways that cancers may be detected,<br />

monitored and treated in the future,” said Roopom Banerjee,<br />

President and CEO of RainDance Technologies. “With the introduction<br />

of our RainDrop System at AACR, we have realised<br />

this vision and officially ushered in the next generation of PCR.<br />

As several of our early customers have demonstrated, we will<br />

soon have absolute answers to many of the most complex<br />

questions in cancer research and tumour progression.”<br />

4


What is digital PCR?<br />

& Why is it important?<br />

Digital PCR, or dPCR, is a significant advancement over conventional polymerase chain reaction methods. It can be used<br />

to clonally amplify and directly quantify nucleic acids including gDNA, cDNA or RNA. The key difference between dPCR and<br />

traditional PCR methods exists in the way they measure nucleic acids amounts, with the dPCR being far more precise.<br />

Traditional PCR carries out one reaction per<br />

sample. In dPCR, the sample is separated<br />

into a large number of partitions and the<br />

reaction is carried out individually within<br />

each partition. As a result, each partition<br />

will contain a negative or positive reaction<br />

(“0” or “1”), respectively.<br />

Mutant<br />

Mutant RainDrop Wild type Mutant Digital PCR System<br />

Nucleic acids are quantified by counting the<br />

Wild type<br />

Empty<br />

Dark<br />

Time (mins)<br />

droplet<br />

droplet<br />

partitions Capable that of contain generating positive more reactions. than a billion reactions in a single day, the<br />

Unlike qPCR, dPCR is not dependent<br />

Create &<br />

PCR<br />

RainDrop Digital PCR System surpasses all existing Sample technologies and<br />

Collect Amplification<br />

on the establishes number of a amplification new performance cycles standard in sensitivity, quantitation, and<br />

Identify & Count<br />

to determine multiplexing. the initial Based amount on RainDance’s the proven picodroplet platform, the<br />

sample, RainDrop eliminating System the delivers reliance absolute on uncertain quantitation of target molecules. The<br />

exponential RainDrop data Digital to quantify PCR System targets, fundamentally thereby providing changes absolute the performance years of of developing droplet-based PCR applications.<br />

quantitation. molecular assays by enabling digital answers across a number of The important RainDrop dPCR system is comprised of two instruments:<br />

applications including low-frequency tumor allele detection, absolute the RainDrop Source which creates and collects the droplets<br />

Since quantitation dPCR is an of ‘end expression point assay’, profiles it does or DNA not require copy numbers, standard and and the RainDrop Sense which individually identifies and counts<br />

curves SNP either. measurement. The method has been shown to be useful for<br />

studying variations in gene sequences - such as detecting rare<br />

mutations, Advantages Copy Number Variants (CNV) and point mutations.<br />

Absolute quantitation is crucial in accurately determining higher<br />

each droplet as it rapidly moves past the laser.<br />

The RainDrop Source runs up to 8 lanes (25 – 50 µl input volume)<br />

in parallel. Essentially it divides the sample between millions of<br />

CNV levels quantifying rare variants below the level where • Detect 1 mutant picoliter amongst droplets. 250,000 This wild-type process is molecules optimised with to a produce lower limit<br />

Superior sensitivity<br />

a single<br />

of detection of 1 in more than 1,000,000<br />

qPCR fails due to competitive amplification of the common DNA. copy reaction per droplet. After PCR is complete, the strip<br />

• Conduct 10 tubes tests or are more attached on the to same the Sense sample chip. using The the system single molecule is designed in<br />

Unprecedented multiplexing<br />

Such new capabilities are vital in understanding causative multi-color detection such a way technique that it is a completely closed tube operation, which<br />

somatic changes and the impact of therapies on cancer<br />

dramatically minimises any chance of cross contamination.<br />

• Optimize the number of PCR reactions based on your sensitivity AND<br />

patients,<br />

Greater<br />

including<br />

study<br />

monitoring<br />

design flexibility<br />

residual disease.<br />

multiplex requirements<br />

Droplet-based digital PCR data looks similar to simple flow<br />

What Closed-tube is the RainDance design RainDrop<br />

digital PCR system?<br />

• Ensure the cytometry highest quality results; data a by two eliminating color fluorescent contamination intensity or carryover signal for<br />

each droplet. RainDrop software enables conversion of data<br />

• Leverage a<br />

The system uses the patented and proven RainStorm microdroplet<br />

technology, and builds on the expertise gained from<br />

into decade cytometry of experience industry with standard the same FCS technology 3.0 format found for in use with<br />

Proven picodroplet platform<br />

RainDance’s established Targeted Sequencing analysis packages. Systems<br />

Wild type<br />

A Billion Reactions. Digital Answers.<br />

The Next-Generation of PCR is Here.<br />

Lowest cost per data point<br />

A graphical summary is shown below:<br />

Fluroscent Intensity (V)<br />

0.8<br />

0.7<br />

0.6<br />

0.5<br />

0.4<br />

0.3<br />

0.2<br />

0.1<br />

0.0<br />

0.0 0.5 1.0<br />

1.5 2.0<br />

• Generate true digital answers with orders of magnitude more data per dollar<br />

Streamlined Workflow<br />

Streamlined Workflow<br />

Sample Create & Collect <strong>Thermo</strong>cycle Identify & Count Analyze<br />

5


The performance advantage of the RainDrop system is driven by<br />

its 10 million droplets per lane to provide the following benefits:<br />

Superior sensitivity<br />

Unprecedented multiplexing<br />

Greater study design flexibility<br />

Detect 1 mutant amongst 250,000 wild-type molecules with a lower limit of<br />

detection of 1 in more than 1,000,000<br />

Conduct 10 tests or more on the same sample using the single molecule<br />

multi-colour detection technique<br />

Optimise the number of PCR reactions based on your sensitivity and<br />

multiplex requirements<br />

Closed-tube design<br />

Ensure the highest quality data by eliminating contamination or carryover<br />

Proven picodroplet platform<br />

Lowest cost per data point<br />

Leverage a decade of experience with the same technology found in<br />

RainDance’s Targeted Sequencing Systems<br />

Uncompromised Results<br />

The RainDrop System shifts the PCR paradigm from a single color per marker to a more scalabl<br />

color and intensity-per-marker method. This novel approach increases sensitivity by generating b<br />

million pico-liter sized droplets per lane, which is a 500 – 10,000x improvement over existing me<br />

encapsulates a single molecule, researchers can quickly determine the absolute number of drop<br />

target DNA and compare that to the amount of droplets with normal, background wild-type DNA<br />

Generate true digital answers with orders of magnitude more data per dollar<br />

6<br />

Uncompromised Results<br />

The RainDrop System shifts the PCR paradigm from a single colour per<br />

marker to a more scalable and precise multi-colour and intensity-per-marker<br />

method. This novel approach increases sensitivity by generating between<br />

1 million and 10 million pico-liter sized droplets per lane, which is a 500<br />

– 10,000x improvement over existing methods. Since each droplet encapsulates<br />

a single molecule, researchers can quickly determine the absolute<br />

number of droplets containing specific target DNA and compare that to the<br />

Uncompromised Results<br />

amount of droplets with normal, background wild-type DNA.<br />

Superior Sensitivity<br />

To assess the lower limit of detection (LLOD) of an EGFR mutation, varying<br />

concentrations of mutant were diluted<br />

Superior<br />

into genomic<br />

Sensitivity<br />

wild-type DNA. The<br />

assay delivered a linear response (R 2 = 0.998) down to 1 mutant amongst<br />

1E-3<br />

250,000 wild-type molecules, with a LLOD of 1 in more than 1,000,000 as<br />

defined by the average of the wild-type only controls plus 3x the standard<br />

1E-4<br />

deviation.<br />

1E-5<br />

Unprecedented Multiplexing<br />

By adjusting concentrations of the probes for each individual assay, 4<br />

5E-7<br />

mutations plus wild-type were measured simultaneously with just VIC and<br />

0.0<br />

FAM fluorophores. Multiplexing can be expanded to higher plex levels, and<br />

0 1 2 3 4<br />

Dilution Number<br />

it enables detection, identification, and measurement of multiple mutations<br />

from a single DNA sample.<br />

Superior Sensitivity<br />

Mutant to Wild-Type Ratio<br />

1E-3<br />

1E-4<br />

1E-5<br />

5E-7<br />

No Template<br />

Controls<br />

The RainDrop System shifts the PCR paradigm from a single color per marker 0.0 to a more scalable and precise multicolor<br />

and intensity-per-marker method. This novel approach increases sensitivity by generating between 1 million and 10<br />

0 1 2 3 4<br />

Dilution Number<br />

million pico-liter sized droplets per lane, which is a 500 – 10,000x improvement over existing methods. Since each droplet<br />

encapsulates a single molecule, researchers can quickly determine To assess the the absolute lower limit of number detection (LLOD) of of droplets an EGFR mutation, containing varying concentrations specific<br />

of mutant were diluted into genomic wild-type The assay delivered a linear response<br />

target DNA and compare that to the amount of droplets with normal, background wild-type DNA.<br />

(R 2 = 0.998) down to 1 mutant amongst 250,000 wild-type molecules, with a LLOD of 1 in<br />

more than 1,000,000 as defined by the average of the wild-type only controls plus 3x the<br />

standard deviation.<br />

Mutant to Wild-Type Ratio<br />

No Template<br />

Controls<br />

To assess the lower limit of detection (LLOD) of an EGFR mutation, varying concentrations<br />

of mutant were diluted into genomic wild-type DNA. The assay delivered a linear response<br />

(R 2 = 0.998) down to 1 mutant amongst 250,000 wild-type molecules, with a LLOD of 1 in<br />

more than 1,000,000 as defined by the average of the wild-type only controls plus 3x the<br />

standard deviation.<br />

System Components<br />

Two benchtop instruments (P/N: 20-04400), each with its own<br />

PC workstation<br />

RainDrop Source (P/N: 20-04401): Creates and collects up<br />

Unprecedented Multiplexing<br />

System 120– Components<br />

110–<br />

Two benchtop wild-type instruments (P/N: 20-04400), each with its own<br />

100–<br />

G12A<br />

PC workstation<br />

VIC Intensity (arb.)<br />

130–<br />

90–<br />

80–<br />

70–<br />

60–<br />

50–<br />

40–<br />

RainDrop Source (P/N: 20-04401): Creates and collects up<br />

to 10 million picodroplets per lane<br />

–30<br />

No PCR<br />

G12C<br />

RainDrop Sense (P/N: 20-04402): Identifies and counts<br />

each picodroplet following PCR amplification<br />

RainDrop data analysis package<br />

30–<br />

G12V G12S<br />

30–<br />

System 10– Features<br />

50 75 100 125 150 175 200 225 250 275 300 325 350 375 400<br />

FAM Intensity (arb.)<br />

Closed tube design<br />

FAM & VIC detection channels<br />

By adjusting concentrations of the probes for each individual assay, 4 mutations plus wild-type were<br />

measured Graphical simultaneously user with just interface VIC and FAM fluorophores. provides Multiplexing intuitive can be operation<br />

expanded to<br />

higher plex levels, and it enables detection, identification, and measurement of multiple mutations<br />

Integrated bar-coding to record all reagents and<br />

from a single DNA sample.<br />

consumables<br />

Industry standard data output format (FCS 3.0)<br />

Standard one year warranty<br />

System Specifications<br />

Unprecedented Multiple<br />

To assess the lower limit of<br />

detection (LLOD) of an EGFR<br />

130–<br />

mutation, varying concentrations<br />

of mutant were diluted<br />

120–<br />

110–<br />

into genomic wild-type<br />

100– wild-type DNA.<br />

The assay 90– delivered a linear<br />

80–<br />

response (R 2 = 0.998) down<br />

70–<br />

to 1 mutant No amongst PCR250,000<br />

60–<br />

wild-type 50– molecules, with<br />

a LLOD 40– of 1 in more than<br />

30–<br />

1,000,000 as defined by the G12V<br />

30–<br />

average of the wild-type only<br />

controls plus 3x the standard<br />

deviation.<br />

By adjusting concentrations of the probes for each individ<br />

measured simultaneously with just VIC and FAM fluoroph<br />

higher plex levels, and it enables detection, identification<br />

from a single DNA sample.<br />

By adjusting concentrations<br />

of the probes for each<br />

individual assay, 4 mutations<br />

plus System wild-type were Specifications<br />

measured<br />

simultaneously Instrument with dimensions just VIC and (Height x<br />

FAM fluorophores. 17 x 10 x 14.6 Multiplexing inches; 43 x 26 x<br />

can be Voltage/Frequency/Power: expanded to higher plex 120-2<br />

levels, Operating and it enables temperature detection, range: 15<br />

identification, Operating and measurement temp fluctuation:


Direct PCR,<br />

amplify without purification<br />

PCR directly from sample tissue • From plant •<br />

From animal • From human • From blood •<br />

Without prior DNA purification


Table 1<br />

35 cycles<br />

Temperature 95 °C 95 °C 62 °C 72 °C 72 °C 10°C<br />

Time 5 min 20 s 20 s 30 s 5 min hold<br />

8


<strong>Thermo</strong>cycler PCR plate: mean evaporation [%]<br />

Mastercycler<br />

pro S<br />

Corner (4 wells) Edge (32 wells) Center (60 wells)<br />

3 2 0<br />

A 45 32 10<br />

B 30 5 1<br />

C 4 3 6<br />

D 49 5 0<br />

E >50* 30 0<br />

+<br />

9


Direct PCR:<br />

Genotyping Transgenic Mice<br />

Acknowledgements<br />

Transgenic mouse<br />

samples were provided by<br />

Dr. Raija Soininen, Biocenter<br />

Oulu Transgenic core facility,<br />

University of Oulu and Dr. Jaana<br />

Vesterinen, Institute of<br />

Biomedicine/Biochemistry,<br />

University of Helsinki.<br />

References<br />

1. Wang, Z. and Storm, D.R.<br />

2006. Extraction of DNA from<br />

mouse tails. BioTechniques<br />

41:410-412.<br />

2. Malumbres, M. et al.<br />

1997. Isolation of High<br />

Molecular Weight DNA for<br />

Reliable Genotyping of Mice.<br />

BioTechniques 22:1114-1119.<br />

3. Wang, Y. et al. 2004. A<br />

novel strategy to engineer DNA<br />

polymerases for enhanced<br />

processivity and improved<br />

performance in vitro. Nucleic<br />

Acids Research 32:1197-1207.<br />

Gene transfer into mice is extensively<br />

used to study the roles of genes in<br />

development, physiology and human<br />

disease. The use of these animals<br />

requires screening for the presence of<br />

the transgene, usually with PCR.<br />

Traditionally, this involves a DNA<br />

isolation step, during which DNA for<br />

PCR analysis is purified from ear, tail or<br />

toe tissues.¹ ² Kits have been developed<br />

that can be used for genotyping of<br />

transgenic mice without prior DNA<br />

purification.<br />

<strong>Thermo</strong> Scientific Phire Animal Tissue<br />

Direct PCR kit is designed for DNA<br />

amplification directly from animal<br />

tissues. Here we present protocols for<br />

transgenic mice genotyping achieved<br />

directly from mouse ear tissues using<br />

the kit. The results obtained with this kit<br />

are extremely robust, with greater yields<br />

as compared to a commercial DNA<br />

purification system used together with a<br />

conventional hot start Taq DNA<br />

polymerase. When combined with<br />

<strong>Thermo</strong> Scientific fast Piko Thermal<br />

Cycler and UTW PCR Plates, PCR<br />

protocols can be completed in as little<br />

as 40 minutes.<br />

Materials and Methods<br />

• Phire Animal Tissue Direct PCR Kit<br />

• 24-well Piko ® Thermal cycler<br />

• Piko PCR Plates<br />

• Frozen transgenic mouse ears<br />

In the first method, two different primer pairs are multiplexed<br />

in one PCR reaction. We also tested a more challenging<br />

genotyping approach where only one primer set is used for<br />

amplification of two different DNA fragments with a large size<br />

difference.<br />

Components 20 μl reaction 50 μl reaction Final Conc.<br />

H 2<br />

O add to 20 μl add to 50 μl<br />

2x Phire Animal<br />

Tissue PCR Buffer<br />

10 μl 25 μl 1x<br />

primer A X μl X μl 0.5 μl<br />

primer B X μl X μl 0.5 μl<br />

Phire Hot Start II<br />

DNA Polymerase<br />

Sample:<br />

Direct protocol<br />

Dilution protocol<br />

0.4 μl 1 μl<br />

-<br />

1 μl<br />

0.5 mm punch<br />

-<br />

2-step protocol 3-step protocol Final Conc.<br />

Cycle step Temp Time Temp Time Cycles<br />

Lysis of cells 98°C 5 min 98°C 5 min 1<br />

Denaturation 98°C 5 s 98°C 5 s<br />

Annealing - - X°C 5 s<br />

Extension 72°C 20 s < 1 kb<br />

20 s/kb >1 kb<br />

72°C 20 s < 1 kb<br />

20 s/kb >1 kb<br />

Final extension 72°C 1 min 72°C 1 min 1<br />

4°C hold 4°C hold<br />

Direct protocol: A 0.5 mm tissue punch was cut using the<br />

Harris Uni-Core puncher and placed directly into a 50μl PCR<br />

reaction. DNA Release Additive was included in the gel loading<br />

dye when analysing PCR products on an agarose gel.<br />

Dilution protocol: A 2 mm punch of mouse ear was placed in<br />

20μl of Dilution Buffer containing 0.5μl of DNARelease Additive.<br />

The samples were incubated at RT for 2 min and then at 98°C<br />

for 2 min. The samples were spun down and 1μl of supernatant<br />

was used as a template in a 20μl PCR reaction. The supernatant<br />

was transferred to a new tube and stored at -20°C if not used<br />

right away.<br />

40<br />

Stability assays: Mouse ear tissues were prepared as<br />

described for dilution protocol. The reactions were either frozen<br />

and thawed for 20 times or stored at -20°C for 1 year before<br />

PCR.<br />

10


Figure 1. Genotyping of transgenic mice<br />

with two primer pairs using the direct<br />

protocol. 0.50 mm tissue punches of 11<br />

individual mice were placed directly into<br />

50 μl PCR reactions. Fragment sizes: 490 bp<br />

(transgenic) and 250 bp (wildtype).<br />

Figure 4. Dilution protocol samples are stable<br />

for long term storage. Samples of mouse<br />

ear M tissues 1 2 were 3 4 incubated 5 6 7 in 8 209 μl 10 of Dilution<br />

Buffer including DNARelease Additive.<br />

11 - M<br />

Dilution protocol samples were subjected to<br />

repeated freezing/thawing (lane 1), stored at<br />

-20°C for one year (lane 2) as described in<br />

Materials and Methods, or used immediately<br />

Biocenter Oulu Transgenic<br />

facility, University of Oulu<br />

Jaana Vesterinen, Institute<br />

medicine/Biochemistry, Un<br />

M + - 1 2 3 + - 1 2 3<br />

of Helsinki.<br />

References<br />

1 Wang, Z. and Storm, D.R<br />

Extraction of DNA from<br />

M 1 2 3 4 5 6 7 8 9 for PCR (lane 3). The fragment sizes were<br />

Figure 4. Dilution protocol samp<br />

900 Figure bp, 1500 1. Genotyping bp and 3200 of bp. transgenic mice tails. BioTechniques 41:4<br />

ble for long term storage. Samp<br />

with two primer pairs using the direct 2. ear Malumbres, tissues were M. incubated et al. in 192<br />

compared protocol. 0.50 to mm a combination tissue punches of a 11<br />

Isolation Buffer including of High DNARelea Molec<br />

individual mice were placed directly into<br />

commercial DNA extraction kit Dilution<br />

50 μl PCR reactions. Fragment sizes: 490 bp Weight protocol DNA samples for Reliabl were<br />

designed (transgenic) for and animal 250 bp tissues (wildtype). and<br />

repeated freezing/thawing (lane<br />

Figure 2.<br />

-20°C<br />

typing Figure 1. for Genotyping one<br />

of Mice.<br />

year of (lane<br />

BioTech<br />

Figure 1.<br />

2) as de<br />

a Taq based hot start DNA polymerase<br />

M 1 in amplification 2 3 4 5 of 6 four 7 8 9 for pairs PCR using (lane the direct 3). protocol.<br />

Materials 22:1114-1119.<br />

transgenic mice and with Methods, two primer or used<br />

Figure 2. Genotyping of transgenic mice<br />

3. Wang, Y. et al. The 2004. fragment A ns<br />

0.5 mm tissue punches of<br />

with M 1 one 2 primer 3 4 pair 5 6 using 7 8 the 9 dilution 10 11 - M DNA M + - fragments. 1 2 3 + - Phire 1 2 3 Animal Acknowledgements<br />

900 bp, 1500 bp and 3200 bp.<br />

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In Other Countries 11


&<br />

Monitoring Tumour<br />

Metastases<br />

Osteolytic Lesions with<br />

Bioluminescence &<br />

MicroCT Imaging<br />

Lim, E., Modi, K., Christensen,<br />

A., Meganck, J., Oldfield, S.,<br />

Zhang, N. . Monitoring Tumor<br />

Metastases and Osteolytic<br />

Lesions with Bioluminescence<br />

and Micro CT Imaging. J.<br />

Vis. Exp. (50), e2775, DOI:<br />

10.3791/2775 (2011).<br />

Disclosures:<br />

The authors are employees<br />

of Caliper Life Sciences who<br />

make the instrument featured in<br />

this article.<br />

Longitudinal imaging is used in pre-clinical<br />

studies to follow the progress of a disease or<br />

measure the effect of a therapeutic. In oncology,<br />

optical methods provide rigorous tools to monitor<br />

tumour growth and deliver precise quantitation<br />

of cell growth or gene expression at each time<br />

point in such a study. Anatomical changes can<br />

be measured using a high resolution technique<br />

like microCT, but for longitudinal imaging a low<br />

X-ray dose must be used to avoid biological<br />

artifacts. Optical and microCT images can be coregistered<br />

to provide a combination of functional<br />

and anatomical data ensuring that maximum<br />

information is extracted from the animal model.<br />

Following intracardiac delivery of MDA-MB-<br />

231-luc-D3H2LN cells to Nu/Nu mice,<br />

systemic metastases developed in<br />

the injected animals. Bioluminescence<br />

imaging using IVIS Spectrum was<br />

employed to monitor the distribution<br />

and development of the tumour cells following<br />

the delivery procedure including DLIT reconstruction to<br />

measure the tumour signal and its location.<br />

Development of metastatic lesions to the bone tissues triggers<br />

osteolytic activity and lesions to tibia and femur were evaluated<br />

longitudinally using microCT. Imaging was performed using<br />

a Quantum FX microCT system with fast imaging and low<br />

X-ray dose. The low radiation dose allows multiple imaging<br />

sessions to be performed with a cumulative X-ray dosage<br />

far below LD50. A mouse imaging shuttle device was used<br />

to sequentially image the mice with both IVIS Spectrum and<br />

Quantum FX achieving accurate animal positioning in both the<br />

bioluminescence and CT images. The optical and CT data sets<br />

were co-registered in 3-dimentions using the Living Image 4.1<br />

software. This multi-mode approach allows close monitoring<br />

of tumour growth and development simultaneously with<br />

osteolytic activity.<br />

Image: MicroCT image<br />

(Quantum FX) confirming bone<br />

degradation in the right tibia.<br />

12


ONE: Cell preparation<br />

1. Caliper provides a range of luciferase expressing cancer cell<br />

lines for pre-clinical research in mouse models.<br />

2. MDA-MB-231-luc-D2H2LN is a human mammary tumour<br />

cell line expressing the luciferase gene which can be used<br />

as an optical indicator of tumourgenesis in vivo. This cell line<br />

is created from a spontaneous lymph node metastasis that<br />

originated from a D3H1 mammary fat pad tumour and is<br />

known to aggressively form metastases.<br />

3. The cells are provided as a pathogen-free frozen culture<br />

which readily grows in standard media with no need for<br />

selection markers.<br />

4. To verify luciferase activity before injection into the animal<br />

a 90% confluent flask is harvested by trypsinisation.<br />

Luciferase activity is measured by dispensing 50,000 cells<br />

in a microtiter plate and performing serial dilution.<br />

TWO: Intracardiac injection of cells in<br />

animals<br />

1. Before injection, animals are anesthetised using 3%<br />

isoflurane.<br />

2. 1-3 million cells are injected in 50µl volume into the left<br />

ventricle. Cell suspension contains 150µg/ml D-Luciferin to<br />

validate injection technique.<br />

3. To confirm a correct intracardiac injection mice are imaged<br />

in the IVIS system. Mice that show BLI signal throughout the<br />

body have been injected correctly. If signal is localised the<br />

cells were not injected correctly in the heart.<br />

THREE: BLI imaging to monitor metastases<br />

1. The imaging shuttle supports the mouse for data capture in<br />

both IVIS Spectrum and the QuantumFX microCT system.<br />

2. The shuttle helps maintain the animal in the same position<br />

for imaging and incorporates fiducial signals to aid in<br />

automatic co-registration. The chamber is connected to the<br />

isofluorance vaporiser to maintain anesthesia.<br />

3. Imaging wizard in the Living Image software 4.1 can be<br />

used to automatically select the exposure time, f-stop and<br />

binning and ensure quantitative data collection.<br />

4. Multiple images can be acquired and compared in<br />

longitudinal studies covering seconds or months depending<br />

on the nature of the experiment.<br />

5. Day 6 post injection of cells, BLI imaging with IVIS Spectrum<br />

shows metastases to the bone in seven out of nine mice.<br />

FOUR: Longitudinal monitoring of<br />

osteolytic lesions using micro CT<br />

1. Mice that showed clear bone metastases visible using<br />

BLI were imaged in the Quantum FX microCT system.<br />

2. The Quantum FX microCT system delivers high quality<br />

images at an X-ray dose low enough to enable true<br />

longitudinal microCT in preclinical studies.<br />

3. Bone degradation in both legs can be longitudinally<br />

monitored using this system in less than a minute.<br />

4. Mice are anesthetised with 3% isoflurane and can be<br />

transferred directly from the IVIS Spectrum system.<br />

5. Two 60mm images are stitched together to capture the<br />

whole animal view.<br />

6. A 30mm FOV is ideal for imaging the knee joint in high<br />

resolution.<br />

7. A single scan with high enough resolution to measure<br />

and quantify bone metastases takes just 18 seconds<br />

and delivers a dose of only 13mGy.<br />

8. Mice were imaged up to 8 times in 36 days with the<br />

microCT system without affecting the tumour growth or<br />

health of the animal.<br />

9. The microCT imaging showed that bone leisions<br />

were visible on Day 10 and progressed very rapidly,<br />

corresponding to the increase in BLI signal.<br />

FIVE: 3D co-registration and analysis of<br />

BLI & microCT<br />

1. Once images have been captured on both the IVIS<br />

spectrum and Quantum FX microCT system data can be<br />

co-registered into a 3D reconstruction.<br />

2. MicroCT images are exported as DICOM files and loaded<br />

into the Living Image 4.1 3D Multi modality tool.<br />

3. In the Living Image Software run the DLIT program to<br />

three dimensionally reconstruct the optical signal.<br />

4. The fiducial tool in the software is used to co-register the<br />

optical BLI signal to the microCT image.<br />

5. Coregistered data sets can be analysed quantitatively to<br />

monitor multiple parameters during disease progression<br />

here we are tracking growth of the metastatic tumour<br />

together with development of the osteolytic lesion in the<br />

bone.<br />

Image Left:<br />

Female Nu/Nu mice were<br />

intracardially injected with<br />

1x10 6 MDA-MB-231-luc-<br />

D3H2LN tumour cells. Bone<br />

metastasis by MDA-MB-231<br />

cells was followed by<br />

bioluminescence, fluorescence<br />

(RediJect 2-DG 750 probe) with<br />

IVIS Spectrum. MicroCT image<br />

showing metastatic lesions<br />

within the bone architecture<br />

and osteolysis was imaged with<br />

Quantum FX microCT imaging<br />

system.<br />

13


References<br />

Jenkins, D.E., Hornig, Y.S.,<br />

Oei, Y., Dusich, J., Purchio, T.<br />

Bioluminescent Human Breast<br />

Cancer Cell Lines that Permit<br />

Rapid and Sensitive in vivo<br />

Detection of Mammary Tumors<br />

and Multiple Metastases<br />

in Immune Deficient Mice.<br />

Breast Cancer Research. 7,<br />

R444-R454 (2005).<br />

Cowey, S., Szafran, A. A.,<br />

Kappes, J., Zinn, K.R., Siegal,<br />

G.P., Desmond, R. A., Kim, H.,<br />

Evans, L., Hardy, R. W. Breast<br />

Cancer Metastasis to Bone:<br />

Evaluation of Bioluminescent<br />

Imaging and MicroSPECT/CT for<br />

Detecting Bone Metastasis in<br />

Immunodeficient Mice. Clinical<br />

and Experimental Metastasis.<br />

24 (5), 389-401 (2007).<br />

Labrinidis, A., Hay, S., Liapis, V.,<br />

Ponomarev, V., Findlay, D.M.,<br />

Evdokiou, A. Zoledronic Acid<br />

Inhibits Both the Osteolytic<br />

and Osteoblastic Components<br />

of Osteosarcoma Lesions in a<br />

Mouse Model. Clinical Cancer<br />

Research. 15(10) (2009).<br />

Kaijzel, E.L., van der Pluijm, G.,<br />

Lowik, C.W.G.M. Whole-Body<br />

Optical Imaging in Animal<br />

Models to Assess Cancer<br />

Development and Progression.<br />

Clinical Cancer Research. 13<br />

(12), 3490-3497 (2007).<br />

Minn, A.J., Kang, Y.,<br />

Serganova, I., Gupta, G.P.,<br />

Giri, D.D., Doubrovin, M.,<br />

Ponomarev, V., Gerald, W.L.,<br />

Blasberg, R., Massague,<br />

J. Distinct Organ-Specific<br />

Metastatic Potential of Individual<br />

Breast Cancer Cells and Primary<br />

Tumors. The Journal of Clinical<br />

Investigation. 115 (1), (2005).<br />

Xu, H, Christensen A., Kuo C.,<br />

Nilson D., Lim E., Whalen J.,<br />

Kim, J.B., Zhang, N., Singh,<br />

R., Oldfield, S., Troy, T., Rice, B.<br />

Co-registration of in vivo Optical<br />

Tomography and Micro-CT for<br />

Longitudinal Studies. WMIC.<br />

#0332B, Kyoto (2010).Lim,<br />

E., Modi, K., Christensen,<br />

A., Meganck, J., Oldfield, S.,<br />

Zhang, N. . Monitoring Tumor<br />

Metastases and Osteolytic<br />

Lesions with Bioluminescence<br />

and Micro CT Imaging. J.<br />

Vis. Exp. (50), e2775, DOI:<br />

10.3791/2775 (2011).<br />

Discussion<br />

Small animal imaging with microCT has been applied to<br />

monitoring osteolytic lesions under pathological conditions,<br />

such as tumour development (Cowey et al. 2007; Labrinidis et<br />

al., 2009). As bioluminescence imaging of luciferase labelled<br />

tumours has been widely adopted pre-clinically, co-registration<br />

of bioluminescence and microCT images provided a better<br />

definition of anatomical location of the bioluminescence signals<br />

(Kaijzel et al. 2007). Previous study with a luciferase labelled<br />

breast tumour cell line MDA-MB-231-luc-D3H2LN showed<br />

metastases to bone and development of osteolytic lesions<br />

following intra-cardiac injection (Jenkins et al 2005; Minn et al.,<br />

2005).<br />

In this study, we explored a longitudinal study of tumour<br />

metastases and osteolytic activity with combinatory optical<br />

and low-radiation-dose microCT imaging with the systemic<br />

MDA-MB-231-luc-D3H2LN metastasis model. We performed<br />

sequential imaging with IVIS Spectrum and Quantum FX<br />

microCT systems and achieved bioluminescence and CT<br />

co-registration with our newly developed Living Image 4.1<br />

software (Heng et al., 2010).<br />

Bioluminescence imaging with IVIS provides non-invasive<br />

detection of the tumour cells with high sensitivity, allowing the<br />

tumours to be detected at early stage. Imaging with Quantum<br />

FX allows accurate 3D reconstruction of the bone lesions and<br />

detailed anatomical information. Quantum FX system offers low<br />

radiation dose imaging, which enables longitudinal monitoring<br />

of disease progression. Correlation of bone lesions and tumour<br />

metastases was depicted in the co-registered image, and the<br />

advantage of multimodality tracking of lesion development<br />

longitudinally was clearly demonstrated.<br />

"Bioluminescence imaging<br />

with IVIS provides noninvasive<br />

detection of the<br />

tumour cells with high<br />

sensitivity, allowing the<br />

tumours to be detected at<br />

early stage. Imaging with<br />

Quantum FX allows accurate<br />

3D reconstruction of the<br />

bone lesions and detailed<br />

anatomical information."<br />

14


Optimal Torque<br />

to Prevent Sample Loss During Cryostorage<br />

Cryopreservation is a common sample storage method for critical scientific research studies.<br />

Sample loss due to evaporation during long-term storage is a common problem that can prove<br />

troublesome or even disastrous. Sample loss can alter the concentration or composition of the<br />

sample, making it impossible to achieve reproducible results.<br />

Brian Hewson, Application<br />

Scientist, <strong>Thermo</strong> <strong>Fisher</strong><br />

Scientific<br />

This study demonstrates how the torque of the storage tube cap affect sample attrition due to<br />

repeated thawing and freezing.<br />

Results<br />

After 20 freeze-thaw cycles, the eight tubes tightened to a<br />

torque of 12 oz-in lost little of their weight (0.01%). The eight<br />

tubes tightened to a torque of 8 oz-in lost approximately 0.02%<br />

of their total weight, while the tubes tightened to a torque of 4<br />

oz-in suffered a loss of just under 0.6%.<br />

Materials<br />

Figure 1. <strong>Thermo</strong> Scientific Matrix 2D Barcoded ScrewTop Tube<br />

1. <strong>Thermo</strong> Scientific Matrix 1.0 mL 2D barcoded, V-bottom,<br />

ScrewTop tubes (Cat. No. 3741).<br />

2. <strong>Thermo</strong> Scientific Matrix1000 µL Single-channel Manual<br />

Pipette (Cat. No. 1204)<br />

3. Quality Biological Eagle’s Minimal Essential Media (Cat<br />

No. 112-016-101)<br />

4. Acros ® Glycerol (Cat. No. 15892-0010)<br />

5. MP Biomedicals Rabbit Serum (Cat. No. 191357)<br />

6. LITL Microplate Defroster<br />

7. Analytical Balance<br />

8. Liquid nitrogen, large container<br />

The sample volumes lost from the caps tightened to 8 oz-in and<br />

12 oz-in are shown to be within the balance tolerances over<br />

the 20 cycles (Figure 2), because sample volumes remained<br />

relatively constant for tubes with caps tightened to both 8<br />

oz-in and 12 oz-in torque, there is no significant advantage to<br />

tightening caps to 12 oz-in torque vs 8 oz-in.<br />

The tubes with caps tightened only to 4 oz-in, however,<br />

were shown to lose a significant sample volume after each<br />

freeze-thaw cycle. Comparing the caps tightened to 4 oz-in to<br />

those tightened to 8 oz-in tubes clearly demonstrated that the<br />

higher torque minimises sample loss over a large number of<br />

freeze-thaw cycles, thereby preserving sample integrity during<br />

cryogenic storage.<br />

Method<br />

Twenty-four Matrix ScrewTop tubes (Figure 1) were filled with<br />

a 1.0 mL solution of Eagle’s Minimal Essential Media with 10%<br />

glycerol and 10% rabbit serum.<br />

Eight tubes each were tightened to a torque of 4, 8, and<br />

12 ounce-inches (2.8, 5.6, and 8.5 Ncm, respectively) and<br />

placed in a -80 °C freezer for 1 hour prior to being placed in a<br />

large container of Vapor Phase Liquid Nitrogen (VPLN) for at<br />

least 2 hours. To facilitate thawing, the tubes were placed on<br />

a microplate defroster that blew air at ambient temperature<br />

across the bottom of the tubes for approximately 1.5 hours.<br />

The tubes were then wiped down to remove condensation,<br />

inspected for crazing and cracking, and weighed. The freezethaw<br />

cycles were repeated for a total of 20 cycles.<br />

Weight (g)<br />

Weight lost by torque as a % of the original volume<br />

0,700%<br />

0,600%<br />

0,500%<br />

0,400%<br />

0,300%<br />

0,200%<br />

0,100%<br />

0,000%<br />

0 5 10 15 20<br />

-0,100%<br />

Thaw cycle<br />

Figure 2. Sample weight loss 4 oz•in 8 oz•in 12 oz•in<br />

15


A targeted A proteomic platform<br />

for Alzheimer's disease<br />

References<br />

1. Querfurth HW, LaFerla<br />

FM. Alzheimer’s disease.<br />

N Engl J Med. 2010 Jan<br />

28;362(4):329-44.<br />

2. Glenner GG, Wong CW.<br />

Alzheimer’s disease: initial<br />

report of the purifcation and<br />

characterization of a<br />

novel cerebrovascular amyloid<br />

protein. Biochem Biophys<br />

Res Commun. 1984 May<br />

16;120(3):885-90.<br />

3. Masters CL, Simms G,<br />

Weinman NA, Multhaup G,<br />

McDonald BL, Beyreuther K.<br />

Amyloid plaque core protein in<br />

Alzheimer disease and Down<br />

syndrome. Proc Natl Acad Sci U<br />

S A. 1985 Jun;82(12):4245-9.<br />

4. Wong CW, Quaranta V,<br />

Glenner GG. Neuritic plaques<br />

and cerebrovascular amyloid<br />

in Alzheimer disease are<br />

antigenically related. Proc<br />

Natl Acad Sci U S A. 1985<br />

Dec;82(24):8729-32.<br />

5. Blennow K, de Leon MJ,<br />

Zetterberg H. Alzheimer’s<br />

disease. Lancet. 2006 Jul<br />

29;368(9533):387-403.<br />

6. Esch FS, Keim PS, Beattie<br />

EC, Blacher RW, Culwell AR,<br />

Oltersdorf T, et al. Cleavage of<br />

amyloid beta peptide during<br />

constitutive processing of its<br />

precursor. Science. 1990 Jun<br />

1;248(4959):1122-4.<br />

7. Seubert P, Vigo-Pelfrey<br />

C, Esch F, Lee M, Dovey H,<br />

Davis D, et al. Isolation and<br />

quantifcation of soluble<br />

Alzheimer’s beta-peptide from<br />

biological fuids. Nature. 1992<br />

Sep 24;359(6393):325-7.<br />

8. Strozyk D, Blennow K,<br />

White LR, Launer LJ. CSF<br />

Abeta 42 levels correlate with<br />

amyloid-neuropathology in<br />

a population-based autopsy<br />

study. Neurology. 2003 Feb<br />

25;60(4):652-6.<br />

9. Blennow K, Hampel H, Weiner<br />

M, Zetterberg H. Cerebrospinal<br />

fuid and plasma biomarkers<br />

in Alzheimer disease. Nat Rev<br />

Neurol. 2010 Mar;6(3):131-44.<br />

10. Portelius E, Westman-<br />

Brinkmalm A, Zetterberg H,<br />

Blennow K. Determination of<br />

beta-amyloid peptide signatures<br />

in cerebrospinal fuid using<br />

immunoprecipitation-mass<br />

spectrometry. J Proteome Res.<br />

2006 Apr;5(4):1010-6.<br />

11. Portelius E, Tran AJ,<br />

Andreasson U, Persson R,<br />

Brinkmalm G, Zetterberg<br />

H, et al. Characterization<br />

of amyloid beta peptides<br />

in cerebrospinal fuid by an<br />

automated immunoprecipitation<br />

procedure followed by mass<br />

spectrometry. J Proteome Res.<br />

2007 Nov;6(11):4433-9.<br />

An exploration for diagnostic & theragnostic markers<br />

Abstract<br />

The main cause of dementia in the aging population is<br />

Alzheimer’s disease (AD). AD is characterised by several<br />

alterations in the brain, such as accumulation of extracellular<br />

plaques in specific brain regions.<br />

The plaques are composed mainly of fibrilary amyloid-β<br />

(Aβ) peptides which are cleavage products from the<br />

amyloid precursor protein (APP). Using IP-King<strong>Fisher</strong>-MS,<br />

which includes the <strong>Thermo</strong> Scientific King<strong>Fisher</strong> mL for<br />

immunoprecipitation, we have discovered a novel degradation<br />

pathway for APP involving the combined action of α-secretase<br />

and β-secretase. The APP pathway might be upregulated<br />

in AD as an attempt to counteract amyloidogenic APP<br />

processing. The IP-King<strong>Fisher</strong>-MS system enables enhanced<br />

throughput and facilitates larger and clinically more relevant<br />

studies.<br />

Introduction<br />

Alzheimer’s disease (AD) accounts for 50–60% of<br />

approximately 35 million dementia cases around the<br />

world (1) . It is a slowly progressive neurodegenerative disorder<br />

which neuropathologically is characterised by the abnormal<br />

accumulation of intraneuronal neurofibrillary tangles and<br />

extracellular plaques in specific brain regions together with<br />

massive neuronal and synaptic degeneration.<br />

In the mid-1980s it was found that plaques are composed<br />

mainly of fibrilary amyloid-β (Aβ) (2-4) . Much of the fibrillar<br />

Aβ found in the plaques consists of the 42 amino acid<br />

form of Aβ (Aβ1-42). Aβ is produced from the amyloid<br />

precursor protein (APP) by sequential cleavage by β- and<br />

γ-secretase in the amyloidogenic APP-processing pathway<br />

(Figure 1a) (5) . In another described degradation pathway, APP<br />

is cleaved in the middle of the Aβ sequence thus precluding the<br />

formation of the neurotoxic Aβ1-42 isoform (6) .<br />

In the early '90s it was shown that Aβ is secreted to<br />

cerebrospinal fluid (CSF) as a soluble peptide (7) . When selective<br />

methods for measuring Aβ1-42 in the CSF were developed, a<br />

marked decrease was evident in AD patients. This reduction in<br />

CSF Aβ1-42 in the CSF from AD patients is believed to reflect<br />

the AD pathology with plaques in the brain acting as sinks, thus<br />

lowering the level of Aβ1-42 in the CSF (8) . Aβ has been the<br />

subject of extensive targeted proteomic studies aimed to follow<br />

the disrupted balance between the production and clearance<br />

of the peptide and to identify diagnostic- and theragnostic<br />

markers (9) . Here we describe the use of the King<strong>Fisher</strong><br />

mL magnetic particle processor as a tool in targeted Aβ<br />

proteomics which enables enhanced throughput and facilitates<br />

larger and clinically more relevant studies.<br />

Material and Methods<br />

Immunoprecipitation (IP) of Aβ CSF is described elsewhere (10) .<br />

Briefly, an aliquot (4 μL1 mg/mL) of the Aβ specific antibody<br />

(6E10,<br />

epitope<br />

4-9, Signet<br />

Laboratories,<br />

Inc., Dedham, MA,<br />

USA) is bounded to<br />

50 μL Dynabeads M-280<br />

sheep anti-mouse according<br />

to the manufacturer’s product<br />

description. After washing, the beads can be used for<br />

immunoprecipitation of human CSF (11) , mouse CSF and brain<br />

tissue (12) , cell media (13) , or human brain tissue (14) followed<br />

by washing and elution with the protocol described in the<br />

following section.<br />

The beads/sample solution (total volume 1mL) is transferred<br />

to a King<strong>Fisher</strong> mL magnetic particle processor (<strong>Thermo</strong><br />

Scientific Matrix storage tubes) which is used for automated<br />

washing and elution of peptides and/or proteins in a 5-step<br />

procedure. First, the magnetic beads/sample solution is<br />

added to the <strong>Thermo</strong> Scientific King<strong>Fisher</strong> mL tube 1. The<br />

following three wash steps (tubes 2–4) were conducted for 10<br />

seconds in 1 mL of 0.025% Tween-20 in PBS, PBS, and 50<br />

mM ammonium hydrogen carbonate (pH 8.0, Riedel-deHaën),<br />

respectively.<br />

Finally, the washed beads are transferred to the last tube (five)<br />

to which 100 μL of 0.5% formic acid (FA) has been added<br />

for elution (30 seconds) of the Aβ peptides. The collected<br />

supernatant is dried in a vacuum centrifuge and re-dissolved<br />

in 5 μl of 0.1% FA in 20% acetonitrile. The Aβ peptides are then<br />

analysed using MALDI-TOF MS and/or nanoflow LC-MS/MS.<br />

Results<br />

Using the King<strong>Fisher</strong> mL for automatic washing and elution<br />

of Aβ, we have to date identified more than 15 different<br />

isoforms of Aβ (10−11) , 10 APP/ Aβ(X-15) isoforms (15) and several<br />

N-terminal fragments of APP (16) in the CSF (see Figure 2a for<br />

a representative mass spectrum from an AD patient). In a first<br />

pilot study, we reported an increase in Aβ1-16 together with<br />

the expected decrease in Aβ1-42 in the CSF from patients with<br />

sporadic AD (17) .<br />

This finding was recently confirmed in another study in the<br />

CSF from patients with sporadic AD as well as the CSF from<br />

patients with familial AD using the King<strong>Fisher</strong> mL for automatic<br />

washing and elution of Aβ isoforms (18) . Recently we showed<br />

that cells that had been treated with a γ-secretase inhibitor,<br />

one of the key enzymes for generating Aβ, produced higher<br />

levels of Aβ1-14, Aβ1-15, and Aβ1-16 (13) . The data presented<br />

showed that APP can undergo a novel processing pathway by<br />

concerted β- and α-secretase cleavages, in addition to the well<br />

established amyloidogenic and non-amyloidogenic pathway,<br />

16


Results<br />

Relative gene expression reproducibility among<br />

reagents, researchers, laboratories and instruments<br />

HeLa cells were transfected with siRNA pools at three<br />

concentrations targeting SEC23A, PPIH and RAC1. After 24<br />

hours RNA was purifed in 96-well format. cDNA synthesis<br />

and qPCR steps were performed at two different sites by<br />

different researchers on various real-time instruments. Relative<br />

gene expression was determined using a ΔΔCq method and<br />

normalised to a Reference gene (PPIB) and to a treatment<br />

control (cells transfected with Non-targeting siRNA pool). For<br />

all gene targets a dose-dependent decrease in gene expression<br />

was observed with increasing concentration of siRNA as<br />

expected (Figure 1). The PikoReal instrument demonstrates a<br />

similar level of knockdown to five other commercial real-time<br />

instruments as shown in Figure 1.<br />

Comparison of <strong>Thermo</strong> Scientific qPCR platform and<br />

another commercially available qPCR platform in a gene<br />

silencing experiment.<br />

Next we compared assay performance and relative gene<br />

expression results using different qPCR reagents with different<br />

chemistries. Solaris assays contain a hybridisation probe<br />

that fluoresces when hybridised to its target sequence, while<br />

TaqMan assays contain a hydrolysis probe that fluoresces<br />

when hydrolysed by the DNA polymerase during amplification.<br />

Solaris reagents and TaqMan reagents were used to amplify<br />

SEC23A, PPIH and PPIB (Reference gene) from the same<br />

System and TaqMan reagents were run on alternative Fast<br />

Real-Time PCR System. Assay performance was assessed<br />

using a dynamic range of five log 10<br />

cDNA concentrations and<br />

calculation of amplification efficiency and r 2 value. Table 3<br />

shows that each assay meets high-performance criteria for<br />

amplification efficiency (100% ± 10%) and r 2 values (≥ 0.995).<br />

For the gene silencing experiment, both sets of reagents give<br />

equally accurate measures of targeted gene knockdown at<br />

three different doses of siRNA. As judged by amplification<br />

efficiency (%) and repeatability (r 2 ), employing MGB and<br />

Superbases technologies into Solaris assays, together with<br />

new generation of personalised PikoReal 96 Real-Time PCR<br />

System and UTW plates, the <strong>Thermo</strong> Scientific solution<br />

proves to be working equivalent to an established commercial<br />

platform.<br />

Conclusion<br />

We offer a product platform for the RT-qPCR workflow in<br />

an RNAi experiment to study relative gene expression. We<br />

demonstrate the reproducibility among reagents, researchers<br />

and laboratories for gene expression results from targeted<br />

gene knockdown using Solaris qPCR Gene Expression<br />

reagents and six real-time instruments including the PikoReal<br />

96 Real-Time PCR System. As expected, the <strong>Thermo</strong> Scientific<br />

solution provides equally accurate measures of relative gene<br />

expression in an RNAi experiment compared to<br />

Life Technologies equivalent platform.<br />

References<br />

1. Livak, K.J. and T.D.<br />

Schmittgen. 2001. Analysis of<br />

Relative Gene Expression Data<br />

Using Real-Time Quantitative<br />

PCR and the 2−ΔΔCT method,<br />

Methods 25(4): 402-8.<br />

2. Jackson, B. and A. Haas.<br />

2010. High Performance<br />

RT-qPCR Using <strong>Thermo</strong> Scientific<br />

Solaris qPCR Gene Expression<br />

Reagents for Accessing Relative<br />

Gene Expression, <strong>Thermo</strong><br />

Scientific Technical Note, http://<br />

www.dharmacon.com/<br />

uploadedFiles/Home/<br />

Resources/Product_Literature/<br />

high-performance-qpcr-usingsolaris-reagents.pdf<br />

3. Haimes, J. and M. Kelley.<br />

2010. Demonstration of a ΔΔCq<br />

Calculation Method to Compute<br />

Relative Gene Expression from<br />

qPCR Data, http://www.dharmacon.com/uploadedFiles/Home/<br />

Resources/Product_Literature/<br />

delta_cq_solaris_tech_note.pdf<br />

PikoReal 96 Real-time PCR System (Site 1)<br />

Applied Biosystems Viia7 Real-time PCR System (Site 1)<br />

PikoReal 96 Applied Real-time PCR Biosystems System (Site 7500 1) Fast Real-time PCR Applied System Biosystems (Site Viia7 1) Real-time PCR Applied System (Site Biosystems 1) Prism 7900HT Real-time PCR System (Site 2)<br />

Applied Biosystems 7500 Fast Real-time PCR System (Site 1) Applied Biosystems Prism 7900HT Real-time PCR System (Site 2)<br />

Agilent Mx3005 Agilent QPCR Mx3005 System (Site QPCR 1) System (Site 1) Roche LightCycler 480 System (Site 2) Roche LightCycler 480 System (Site 2)<br />

Relative SEC23A expression normalized to siNTC<br />

1.4<br />

1.2<br />

1.0<br />

0.8<br />

0.6<br />

0.4<br />

0.2<br />

0.0<br />

SEC23A relative expression average biological and<br />

PPIH relative expression average biological and<br />

RAC1 relative expression average biological and<br />

SEC23A technical relative replicates expression average biological and technical replicates PPIH relative expression average technical biological replicates and<br />

technical replicates 1.4<br />

technical 1.4 replicates<br />

1.4<br />

1.2<br />

1.4<br />

1.2<br />

1.4<br />

1.2<br />

1.0<br />

1.0<br />

1.2<br />

1.2<br />

1.0<br />

0.8<br />

0.6<br />

0.8<br />

0.6<br />

0.4<br />

0.2<br />

1.0<br />

0.8<br />

0.6<br />

0.8<br />

0.6<br />

0.4<br />

0.2<br />

1.0<br />

0.8<br />

0.6<br />

0.0<br />

0.0<br />

100 nM<br />

0.4<br />

10 nM 1 nM 100 nM 10 nM 1 nM 100 nM 10 nM 1 nM 100 nM<br />

0.4<br />

10 nM 1 nM 100 nM 10 nM 1 nM 100 nM 10 nM 1 nM<br />

0.4<br />

SEC23A siNTC PPIH siNTC RAC1 siNTC<br />

Relative SEC23A expression normalized to siNTC<br />

Relative PPIH expression normalized to siNTC<br />

Relative PPIH expression normalized to siNTC<br />

Relative RAC1 expression normalized to siNTC<br />

RAC1 relative expression average biological and<br />

technical replicates<br />

r 2 value<br />

Efficiency<br />

Solaris TaqMan Solaris TaqMan<br />

SEC23A 0.999 0.998 90.6% 94.0%<br />

PPIH 0.999 0.999 99.3% 94.0%<br />

PPIB 0.999 0.999 91.2% 96.5%<br />

0.2<br />

0.2<br />

Figure 1. RT-qPCR reproducibility among reagents, researchers, laboratories and instruments.<br />

0.2<br />

Table 3.<br />

siRNA 0.0 targeting SEC23A, PPIH, RAC1 and a non-targeting control (siNTC) were 0.0 transfected into He La cells at 100, 10 and 1 nM final 0.0 Comparison of <strong>Thermo</strong> Scientific Solaris<br />

concentrations. 100 cDNA nM synthesis 10 nM and 1 nM qPCR steps 100 nM were 10 performed nM 1 at nMtwo geographically 100 nM separated 10 nM laboratories 1 nM by 100 different nM researchers 10 nM 1 nM reagents 100 nM and 10 TaqMan nM 1 reagents nM 100 in assay nM 10 nM 1 nM<br />

cDNA on six generated real-time instruments. the SEC23A above Relative RNAi gene experiment. expression siNTC was determined Reactions using a ΔΔCq methos PPIH normalised to PPIB Reference siNTC gene and<br />

performanceRAC1 siNTC<br />

using treatment Solaris control. reagents Error bars were are standard run on PikoReal deviations from 96 biological Real-Time triplicates. PCR<br />

Relative RAC1 expression normalized to siNTC<br />

17 25


Sample Preparation<br />

PCR & qPCR<br />

Visualisation & Analysis<br />

Cloning & Delivery


Solution for RT-qPCR<br />

Relative Gene Expression Analysis<br />

Pak Yang Chum*, Amanda Haas** and Melissa Kelley**<br />

*<strong>Thermo</strong> <strong>Fisher</strong> Scientifc, Vantaa, Finland **<strong>Thermo</strong> <strong>Fisher</strong> Scientifc, Lafayette, CO<br />

In this Technical Note, we demonstrate the utility of <strong>Thermo</strong> Scientific<br />

reagents, consumables and real-time instruments for the<br />

RT-qPCR workflow in an RNA interference (RNAi) experiment to<br />

study relative gene expression. We show reproducibility among<br />

reagents, researchers and laboratories for gene expression<br />

results from targeted gene knockdown using Solaris qPCR<br />

Gene Expression reagents and the PikoReal 96 Real-Time<br />

PCR System as well as five commercially available real-time<br />

instruments. We also illustrate that the same gene expression<br />

results are obtained using <strong>Thermo</strong> Scientific qPCR reagents<br />

and instrument, and another commercially available platform.<br />

Materials and Methods<br />

Cell Culture and siRNA Transfections<br />

HeLa cells were cultured under recommended media conditions<br />

and plated at a density of 10,000 cells per well in 96-well<br />

plates 24 hours before transfection. Cells were transfected<br />

with <strong>Thermo</strong> Scientific ON-TARGET plus SMARTpool reagents<br />

targeting SEC23A, RAC1, PPIH and PPIB at 100, 10 and 1<br />

nM final concentrations using <strong>Thermo</strong> Scientific DharmaFECT<br />

1 transfection reagent at optimised concentration (0.2 μL/<br />

well). Untreated (UT), lipid only (MT) and ON-TARGETplus<br />

Non-Targeting siRNA Pool controls were included. Each<br />

transfection or treatment was performed in triplicate wells<br />

(biological replicates) and cells were harvested 24 hours<br />

post-transfection.<br />

RNA purification and RT-qPCR<br />

RNA was isolated using a 96 Total RNA Isolation System.<br />

cDNA synthesis was performed on a <strong>Thermo</strong> Scientific Piko<br />

Thermal Cycler using <strong>Thermo</strong> Scientific Maxima First Strand<br />

cDNA Synthesis Kit for RT-qPCR according to manufacturer’s<br />

protocol with 100 ng of total RNA in 20 µL reaction.<br />

qPCR experiments were carried out on six different instruments<br />

in three technical replicates of 10 or 12.5 μL reaction volumes<br />

using 1 µL of cDNA. Reactions using Solaris qPCR Gene<br />

Expression assays [SEC23A, PPIB , RAC1 and PPIH] and<br />

Solaris qPCR Gene Expression Master Mixes were run with<br />

the following cycling parameters: DNA polymerase activation<br />

at 95°C for 15 min × 1 cycle; denaturation at 95°C for 15 sec,<br />

annealing/extension at 60°C for 1 min × 40 cycles. Reactions<br />

using TaqMan® Gene Expression assays [SEC23A, PPIB and<br />

PPIH] and TaqMan Gene Expression Master Mix were run on<br />

an alternative Real-Time PCR System (96-well format) using<br />

manufacturer’s recommended cycling protocol. Performance<br />

of Solaris and TaqMan assays were evaluated by amplification<br />

of five 10-fold dilutions of cDNA template with the following<br />

acceptable high performance: r 2 value ≥ 0.995 and amplification<br />

efficiency = 100% ± 10% [efficiency (%) = 10 -1/slope - 1 ].<br />

Relative gene expression analysis calculation using a<br />

ΔΔCq method<br />

Table 1 summarises the relative gene expression analysis<br />

calculation using a ΔΔCq method for experiments with both<br />

biological and technical replicates. First, Cq values of technical<br />

replicates were averaged (Step 1). Then, ΔCq was calculated<br />

by normalising Cq (Target gene) to Cq (Reference gene, PPIB;<br />

Step 2). Next, average ΔCq values were calculated for the<br />

treatment control (here Non-targeting siRNA pool; Step 3).<br />

ΔΔCq was then calculated by normalising ΔCq from Step<br />

2 to average ΔCq in Step 3. Relative gene expression was<br />

calculated (Step 5) as 2-ΔΔCq (Livak and Schmittgen, 2001)<br />

before averaging and calculating the standard deviation (Step<br />

6). For another example on calculating relative gene expression<br />

using a ΔΔCq method for experiments with biological<br />

replicates, see Haimes and Kelley, Technical Note.<br />

Table 1: Overview of steps<br />

involved in expression<br />

analysis calculation using<br />

ΔΔCq Method.<br />

Step 1 Step 2 Step 3 Step 4 Step 5 Step 6<br />

Average Average<br />

siRNA<br />

Biological Technical<br />

treatment<br />

Replicates Replicates Cq Average Average<br />

Average<br />

∆∆Cq ∆∆Cq ∆∆Cq<br />

Cq<br />

TAR Cq<br />

REF<br />

∆Cq<br />

Cq<br />

TAR-REF<br />

∆∆Cq<br />

∆Cq ∆Cq-Avg. ∆Cq<br />

(x nM)<br />

siNTC<br />

Expression Expression Expression<br />

StdDev<br />

i 33.6<br />

26.3<br />

1 ii 33.0 33.3 26.4 26.4 6.9<br />

4.7 0.039<br />

iii 33.2 26.5<br />

i 33.3<br />

26.7<br />

Target 2 ii 33.6 33.6 27.0 26.9 6.7 N/A 4.4 0.046 0.046 0.006<br />

iii 33.9 27.0<br />

i 33.2<br />

26.9<br />

3 ii 33.4 33.4 26.8 26.9 6.5 4.3 0.052<br />

iii 33.8 27.0<br />

i 28.9<br />

26.5<br />

1 ii 29.0 29.0 26.7 26.7 2.3<br />

0.1 0.960<br />

iii 29.0 26.7<br />

Nontargeting<br />

i 29.1<br />

27.0<br />

control<br />

2 ii 29.1 29.2 27.0 27.1 2.1 2.3 -0.1 1.103 1.002 0.087<br />

(siNTC)<br />

iii 29.3 27.2<br />

i 29.9<br />

27.5<br />

3 ii 29.9 29.9 27.6 27.5 2.3 0.1 0.945<br />

iii 29.8 27.5<br />

Step 1<br />

Step 2<br />

Step 3<br />

Average technical replicates<br />

Calculate ∆Cq (Target-Reference)<br />

Average ∆Cq biological replicates Non-targeting<br />

control<br />

Step 4 Calculate ∆∆Cq [∆Cq (from step 2)<br />

-Average ∆Cq (from step 3)<br />

]<br />

Step 5 ∆∆Cq Expression (2 -∆∆Cq )<br />

Step 6<br />

Average biological replicates ∆∆Cq expression and<br />

calculate standard deviation<br />

24


Figure 1 (a)<br />

Figure 1 (a)<br />

Figure 1 (b)<br />

Figure 1 (b)<br />

Figure 2<br />

Cell Membrane<br />

Full Length APP<br />

Cell Membrane<br />

Full Length APP<br />

ß-sAPP<br />

ß-secretase<br />

C99 CTF<br />

ß-sAPP<br />

ß-secretase<br />

C99 CTF<br />

Aß 1-42 Aß 1-42 y-secretase y-secretase<br />

Aß1-40 Aß1-40<br />

Full Length APP<br />

Aß 1-39<br />

Aß1-38<br />

Aß 1-37<br />

Aß 1-34<br />

Aß 1-33<br />

Aß 1-20<br />

Aß 1-19<br />

Aß 1-18<br />

Aß 1-17<br />

Aß 1-39<br />

Aß1-38<br />

Aß 1-37<br />

Aß 1-34<br />

Aß 1-33<br />

Aß 1-20<br />

Aß 1-19<br />

Aß 1-18<br />

Aß 1-17<br />

ß-sAPP<br />

ß-secretase<br />

C99 CTF<br />

Full Length APP<br />

Aß 1-16 α-secretase<br />

ß-sAPP<br />

Aß 1-15<br />

ß-secretase<br />

Aß 1-14<br />

Aß 1-16 α-secretase<br />

Aß 1-15<br />

Aß 1-14<br />

releasing several short Aβ isoforms (Aβ1-16 down to Aβ1-14,<br />

see Figure 1b). We have also verified this novel APP-degrading<br />

pathway in mice (12) and dogs (19) .<br />

Several drug candidates, including inhibitors of β-secretase<br />

and γ-secretase, anti-aggregation agents and Aβ<br />

immunotherapy, are currently being evaluated in clinical trials.<br />

In a phase II clinical trial, 35 individuals with mild to moderate<br />

AD were randomised to placebo or LY450139 (100 or 140 mg)<br />

and the CSF were collected at baseline and after 14 weeks of<br />

treatment.<br />

Using IP-King<strong>Fisher</strong>-MS, we showed that the CSF levels of<br />

Aβ1-14, Aβ1-15 and Aβ1-16 increased dose-dependently,<br />

showing that these shorter isoforms indeed are theragnostic<br />

markers to detect biochemical effects on APP processing in AD<br />

Figure 2. Representative mass spectra from an AD patient<br />

displaying the Aβ isoform pattern before treatment (a)<br />

and after treatment with 140 mg of the γ-secretase<br />

inhibitor LY450139 (b). The mass spectrometric signal of<br />

Aβ1-14/15/16 increased while Aβ1-34 decreased. The<br />

C99 CTF<br />

longer isoforms, including Aβ1-17, Aβ1-40, and Aβ1-42,<br />

were unaffected by the treatment.<br />

patients treated with LY450139 even at doses that do not affect<br />

Aβ1-42 and Aβ1-40 (Figure 2a-b) (20) .<br />

Conclusion<br />

Using IP-King<strong>Fisher</strong>-MS, we have discovered a novel<br />

degradation pathway for APP involving the combined action<br />

of α-secretase and β-secretase. This pathway might be<br />

upregulated in AD as an attempt to counteract amyloidogenic<br />

APP processing. Further, through its reproducibility and<br />

capacity of handling many samples at the same time, the<br />

King<strong>Fisher</strong> mL has enabled enhanced throughput and<br />

facilitates larger and clinically more relevant studies, including<br />

clinical trials on novel AD therapies. Thus, IP-King<strong>Fisher</strong>-MS<br />

is an important tool to monitor APP/Aβ processing directly<br />

in man, to follow pathogenic processes and discover<br />

theragnostic markers.<br />

12. Portelius E, Zhang B,<br />

Gustavsson MK, Brinkmalm<br />

G, Westman-Brinkmalm A,<br />

Zetterberg H, et al. Effects of<br />

gamma-secretase inhibition on<br />

the amyloid beta isoform pattern<br />

in a mouse model of Alzheimer’s<br />

disease. Neurodegener Dis.<br />

2009;6(5-6):258-62.<br />

13. Portelius E, Price E,<br />

Brinkmalm G, Stiteler M, Olsson<br />

M, Persson R, et al. A novel<br />

pathway for amyloid precursor<br />

protein processing. Neurobiol<br />

Aging. 2009 Jul 13.<br />

14. Portelius E, Bogdanovic<br />

N, Gustavsson MK, Volkmann<br />

I, Brinkmalm G, Zetterberg<br />

H, et al. Mass spectrometric<br />

characterisation of brain<br />

amyloid beta isoform signatures<br />

in familial and sporadic<br />

Alzheimer’s disease. Acta<br />

Neuropathol. 2010 Apr 24.<br />

15. Portelius E, Brinkmalm<br />

G, Tran AJ, Zetterberg H,<br />

Westman-Brinkmalm A,<br />

Blennow K. Identifcation of novel<br />

APP/Abeta isoforms in human<br />

cerebrospinal fuid. Neurodegener<br />

Dis. 2009;6(3):87-94.<br />

16. Portelius E, Brinkmalm<br />

G, Tran A, Andreasson U,<br />

Zetterberg H, Westman-<br />

Brinkmalm A, et al. Identifcation<br />

of novel N-terminal fragments<br />

of amyloid precursor protein in<br />

cerebrospinal fuid. Exp Neurol.<br />

2010 Jun;223(2):351-8.<br />

17. Portelius E, Zetterberg H,<br />

Andreasson U, Brinkmalm G,<br />

Andreasen N, Wallin A, et al. An<br />

Alzheimer’s disease-specifc<br />

beta-amyloid fragment<br />

signature in cerebrospinal<br />

fuid. Neurosci Lett. 2006 Dec<br />

6;409(3):215-9.<br />

18. Portelius E, Andreasson U,<br />

Ringman JM, Buerger K, Daborg<br />

J, Buchhave P, et al. Distinct<br />

cerebrospinal fuid amyloid beta<br />

peptide signatures in sporadic<br />

and PSEN1 A431E-associated<br />

familial Alzheimer’s disease.<br />

Mol Neurodegener. 2010;5:2.<br />

19. Portelius E, Van Broeck B,<br />

Andreasson U, Gustavsson<br />

MK, Mercken M, Zetterberg<br />

H, et al. Acute effect on the<br />

Abetaisoform pattern in CSF<br />

in response to ζ-secretase<br />

modulator and inhibitor<br />

treatment in dogs. Journal of<br />

Alzheimer´s disease. 2010;In<br />

press.<br />

20. Portelius E, Dean RA,<br />

Gustavsson MK, Andreasson U,<br />

Zetterberg H, Siemers E, et al. A<br />

novel Abeta isoform pattern in<br />

CSF refects gamma-secretase<br />

inhibition in Alzheimer<br />

disease. Alzheimers Res Ther.<br />

2010;2(2):7.<br />

17


genomics<br />

workflow


Sample Preparation<br />

DNA/RNA Purification kits • Incubated Shakers<br />

Serological Pipettes • Culture Media Reagents and<br />

Consumables • Filtration Systems • Homogenisers<br />

BioReagents • Centrifuges • Sample Storage Tubes,<br />

Plates and Seals • Freezers • Automated Extraction<br />

Systems • General Labware<br />

DNA/RNA<br />

Extraction Kits<br />

GeneJET Genomic DNA Purification<br />

Kits are designed for rapid and efficient<br />

purification of high quality genomic<br />

DNA from various mammalian cell<br />

culture and tissue samples, whole<br />

blood, bacteria and yeast.<br />

Storage Tubes<br />

<strong>Thermo</strong> Scientific has a complete range<br />

of sample storage tubes. Depending on<br />

your storage needs, our Nunc, Matrix<br />

and ABgene products cover all the<br />

requirements for long and short term<br />

storage. They are manufactured from<br />

low DNA binding material and are DNA/<br />

RNA and pyrogen free.<br />

Automated<br />

DNA/RNA Extraction<br />

The <strong>Thermo</strong> Scientific Kingfisher<br />

offers flexible automation options for<br />

magnteic bead based extraction of<br />

DNA, RNA, Protein and Cells. Get<br />

extemely high yield and quality results<br />

from a varitety of starting materials.<br />

Incubator/Shaker<br />

<strong>Thermo</strong> Scientific MaxQ family of Incubator/<br />

shakers is a versatile range that include<br />

incubated and refrigerated units. With<br />

exceptional innovation, reliability and<br />

accuracy, <strong>Thermo</strong> Scientific MaxQ Shakers,<br />

Platforms, and accessories are the optimal<br />

choice for a busy lab.<br />

Cold Storage<br />

Solutions<br />

<strong>Thermo</strong> Scientific TSU Series delivers<br />

ultimate protection and optimum capacity<br />

for your most critical samples. Built<br />

specifically to protect the most critical<br />

samples, the new TSU Series achieve<br />

outstanding thermal performance, safety<br />

and security through state-of-the-art<br />

engineering.<br />

Cell Lysis Reagent<br />

The preparation of good nuclear protein extracts<br />

is central to the success of many gene<br />

regulation studies. <strong>Thermo</strong> Scientific Pierce<br />

NE-PER Nuclear & Cytoplasmic Extraction Kit<br />

enables stepwise lysis of cells that generates<br />

both functional cytoplasmic and nuclear<br />

protein fractions in less than 2 hours.


PCR & qPCR<br />

Thermal Cyclers • Reverse Transcription (RT) Kits<br />

Plates,Tubes & Strips • DNA Polymerases • dNTPs<br />

Sealing Film • PCR Hoods • Real-time PCR • qPCR<br />

Polymerases • Direct PCR kits • Digital PCR • Master Mixes<br />

Real-time<br />

PCR Systems<br />

<strong>Thermo</strong> Scientific PikoReal Real-Time PCR<br />

System offers excellent qPCR performance<br />

in a tiny box - it's light, quiet and has the<br />

smallest footprint in the market. Real-Time,<br />

Any Time - Now within your reach!<br />

High Fidelity<br />

Polymerase<br />

Phusion Hot Start High-Fidelity DNA<br />

Polymerase is a highly accurate hot<br />

start DNA polymerase. It has now been<br />

upgraded to allow the use of an even<br />

wider range of primers, including those<br />

with low melting temperatures.<br />

Thermal Cyclers<br />

Reproducibility, specificity and speed –<br />

these are the requirements for PCR in any<br />

application. The Eppendorf Mastecycler pro<br />

is unparalleled in its ability to fulfil all these<br />

requirements. As leaders in innovation,<br />

Eppendorf has designed a thermal cycler to<br />

meet the demands of a modern molecular<br />

laboratory. The Mastercycler pro S is the<br />

solution with its vapo.protect technology.<br />

PCR & qPCR Plastics<br />

<strong>Thermo</strong> Scientific ABgene PCR & qPCR<br />

plastics are designed, manufactured and<br />

tested to ensure optimal PCR performance.<br />

The ABgene range includes plates, tubes<br />

and adhesive seals for both PCR and qPCR.<br />

Direct PCR Kits<br />

Direct PCR allows amplification of DNA<br />

directly from different types of starting<br />

materials without prior DNA purification.<br />

A tiny amount of source material is used<br />

directly in the PCR reaction without any<br />

purification steps, allowing savings in<br />

both time and money.<br />

Digital PCR<br />

Capable of generating more than a billion<br />

reactions in a single day, the RainDrop<br />

Digital PCR System surpasses all existing<br />

PCR technologies and establishes a<br />

new performance standard in sensitivity,<br />

quantitation, and multiplexing.


Visualisation & Analysis<br />

Spectrophotometers • UV/VIS Microplate Readers<br />

Gel Documentation/Imaging System • UV/Vis<br />

Transilluminators • Power Supplies • Horizontal Gel<br />

Systems • BioReagents • DNA Markers • Northern/<br />

Southern Transfer • Cellulose Membranes • Labelling<br />

Kits • RNA Interference Kits<br />

Micro-Volume<br />

Analysis<br />

The <strong>Thermo</strong> Scientific NanoDrop range<br />

of spectrophotometers make DNA/RNA<br />

quantitation faster and easier. The patented<br />

sample retention technology, uses as little<br />

as 0.5µl of sample and detection is as easy<br />

as pipette, read and wipe.<br />

Gel Documentation<br />

Systems<br />

Gel documentation is an integral part of<br />

any molecular laboratory. Whether your<br />

application is fluorescence imaging of<br />

stained DNA gels or chemiluminescence<br />

of western blots; UVItec offers a<br />

complete range of gel documentation<br />

solutions to suit all your applications.<br />

Electrophoresis<br />

Supplies<br />

The <strong>Thermo</strong> Scientific Owl Series provides<br />

electrophoresis solutions for nucleic acid<br />

separation, protein separation, sequecing<br />

and electro-blotting. The series ranges from<br />

small to large tanks, combs and power<br />

supplies<br />

Microplate<br />

Spectrophotometry<br />

<strong>Thermo</strong> Scientific Multiskan GO UV/Vis microplate<br />

spectrophotometer offers free wavelength<br />

selection for both 96 & 384 well plates and various<br />

types of cuvettes. It features a broad wavelength<br />

range including UV; pathlength correction and fast<br />

reading speed, making it an ideal tool for virtually<br />

any photometric research application, including<br />

DNA, RNA and protein analysis.<br />

DNA/RNA Ladders<br />

<strong>Thermo</strong> Scientific Fermentas GeneRuler<br />

DNA Ladder Mix is recommended for<br />

sizing and approximate quantification of<br />

a wide range of double-stranded DNA on<br />

agarose gels.<br />

Cuvette<br />

Spectrophotometry<br />

The GENESYS 10S spectrophotometer<br />

series offers excellent value, providing<br />

high-performance, reliability, ease-of-use<br />

and a low cost of ownership. It utilises a<br />

high-intensity xenon lamp and dual-beam<br />

optical geometry to deliver unsurpassed<br />

data quality.


Cloning & Delivery<br />

Electroporators • Gel Extraction Kits • Enzymes • Protein<br />

Expression Vectors • Transfection Reagents • Restriction<br />

Enzymes • Cloning Kits • DNA Ligation Kits • Bacterial Media<br />

Shaking Incubators<br />

Electroporators<br />

The Eppendorf Eporator is a compact<br />

instrument designed for fast and controlled<br />

electroporation of bacteria, yeasts and<br />

other micro-organisms. It is easy to use<br />

with intuitive operation and user friendly<br />

programming.<br />

Cloning Kits<br />

CloneJET PCR Cloning Kit is an<br />

advanced positive selection system for<br />

high efficiency cloning of PCR products<br />

generated with any thermostable<br />

DNA polymerase. The kit is ideal for<br />

phosphorylated or non-phosphorylated<br />

DNA fragments.<br />

Gel Extraction Kits<br />

<strong>Thermo</strong> Scientific Fermentas GeneJET<br />

Gel Extraction Kits are designed for<br />

rapid and efficient purification of DNA<br />

fragments from standard or low-melting<br />

point agarose gels run in either TAE or<br />

TBE buffer.<br />

Protein<br />

Expression Vectors<br />

The Pierce Human in vitro Protein Expression<br />

System is a method for expressing<br />

proteins from DNA or mRNA templates in a<br />

cell-free solution. This small-scale expression<br />

method makes it easy to express numerous<br />

mutant variants simultaneously. Additionally,<br />

expression in vitro enables synthesis of toxic<br />

proteins that can not be produced in live cells.<br />

Restriction enzymes<br />

<strong>Thermo</strong> Scientific Fermentas<br />

FastDigest enzymes are an advanced<br />

line of restriction enzymes for rapid<br />

DNA digestion. All FastDigest enzymes<br />

are 100% active in the universal<br />

FastDigest buffers and are able to<br />

digest DNA in 5-15 minutes.<br />

Transfection Reagents<br />

TurboFect in vivo Transfection Reagent is<br />

a sterile solution of a cationic polymer in<br />

water. The polymer forms compact, stable,<br />

positively charged complexes with DNA.<br />

These complexes protect DNA from degradation<br />

and facilitate gene delivery in vivo.


NEW!<br />

Reverse transcription enzymes<br />

derived by in vitro evolution of proteins<br />

Reverse Transcriptases are RNA- and DNA-dependent DNA polymerases that have 5’-3’ DNA polymerisation<br />

activity and RNase H activity (specific to RNA in RNA-DNA hybrids). Commonly used in Reverse Transcription<br />

Polymerase Reaction (RT-PCR) and Reverse Transcription –Real Time Polymerase Chain Reaction (RT-qPCR),<br />

these enzymes allow PCR analysis of RNA.<br />

The most widely used wild-type enzymes are MMuLV RT<br />

(Moloney Murine Leucaemia Virus) and AMV RT (Avian Mieloblastosis<br />

Virus). With technology progressing, there is strong<br />

demand for improved thermostability, the ability to amplify<br />

larger fragments and increased sensitivity. There are a number<br />

of ways to obtain enzymes that address these requirements<br />

for more advanced technology and they include; searching<br />

for new organisms in unusual habitats, rational design of the<br />

enzyme or in vitro evolution of the protein for desired feature.<br />

Searching for new organisms in unusual habitats is luck<br />

dependent and could be time-consuming and costly; rational<br />

design depends on the current understanding and molecular<br />

knowledge of the enzyme; alternatively, in vitro evolution allows<br />

selection of mutants with the desired features from a large<br />

library of potential candidates. The method of in vitro evolution<br />

was the basis for <strong>Thermo</strong> Scientific’s patent pending technology:<br />

Compartmentalised Ribosome Display (CRD).<br />

Figure 2a - Increased<br />

thermostability of Maxima ®<br />

when compared to competitor<br />

enzymes. Figure 2a & 2b – Both<br />

figures show cDNA synthesis<br />

incorporating radioactive label<br />

(dATP[ 33 P]) using 1µg of Ambion<br />

RNA Millennium markers (polyA<br />

tailed) with oligo(dT) 18<br />

primer at<br />

different temperatures. Reaction<br />

products were loaded on 1%<br />

NaOH alkaline agarose gel.<br />

Figure 1 - RTs were incubated<br />

at 50°C in 1x reaction mixture.<br />

Enzyme activity was determined<br />

in a standard activity assay at<br />

indicated time points and time is<br />

displayed in minutes.<br />

3 rd generation-multiple<br />

mutations through the gene<br />

2 nd generation-point mutation<br />

RNase H minus<br />

1 st generation-wild type<br />

Using this technology, two new Reverse Transcriptases were<br />

identified; Maxima ® Reverse Transcriptase and RevertAid TM<br />

Premium Reverse Transcriptase. Upon sequencing, Maxima ®<br />

was found to have 11 mutations and RNase H plus activity,<br />

whereas RevertAid TM Premium was found to have 12 mutations<br />

and RNase H minus activity. In addition, both enzymes were<br />

found to have dramatically increased thermal stability.<br />

Figure 1 demonstrates the increased thermal stability of these<br />

3rd generation enzymes and even after 240min at 50°C both<br />

enzymes retain approximately 95% activity. When compared to<br />

other competitor enzymes, Maxima ® and Revertaid TM display<br />

the widest temperature range and highest thermostability<br />

(Figure 2a & b).<br />

Figure 2b- Increased thermostability of Revertaid TM when compared to competitor enzymes.<br />

Increased thermostability comes with a number of practical<br />

benefits. Firstly, Maxima ® and Revertaid TM Premium are<br />

functional at higher temperatures allowing for higher<br />

specificity of gene specific priming when RNA is rich in<br />

secondary structure. Secondly, Maxima ® and Revertaid TM<br />

Premium are more stable during the reaction resulting in higher<br />

yields and higher sensitivity of first strand cDNA synthesis kit.<br />

Lastly, Maxima ® and Revertaid TM Premium can withstand the<br />

sometimes variable conditions associated with handling and<br />

storage; this also makes Maxima ® and Revertaid TM Premium<br />

more reliable and less likely to have activity issues if handled<br />

poorly.<br />

In addition to increased thermostability both Maxima ® and<br />

Revertaid TM Premium are able to synthesise large cDNA<br />

fragments. In a two-step RT-PCR amplification, Maxima ® is<br />

able to amplify a fragment of up to 20.2kb which is double the<br />

fragment size amplifiable by competitor enzymes (Figure 3).<br />

26


Figure 3 - Two-step RT-PCR with Maxima ® RT.<br />

Amplification of targets up to 20 kb in two step RT-PCR<br />

1µg of total RNA from Jurat cells (1 and 2) or total RNA from<br />

mouse (3 and 4) were used in a reverse transcription reaction<br />

with Maxima ® Reverse Transcriptase and other RTs in the<br />

optimal conditions for each enzyme, Synthesised cDNA was<br />

used as a template in PCR with the Long PCR Enzyme MIX<br />

(#K0181) and primers specific for different genes:<br />

1. 6.8 kb POLE (human polymerase gene),<br />

2. 9.4 kb FBN1 (human fibrillin 1 gene).<br />

3. 13.3 kb Dmd (mouse dystrophin gene),<br />

4. 20.2 kb Neb (mouse nebulin gene),<br />

M GeneRuler 1 kb Plus DNA ladder (#SM1331).<br />

Increased reaction rate is another feature of Maxima ® RT.<br />

When compared to competitor enzymes, Maxima ® RT<br />

was the only enzyme (from those tested) able to amplify<br />

a 7kb fragment in 5 minutes. When various RNA<br />

concentrations were used to measure the sensitivity of<br />

a variety of reverse transcriptase enzymes, RevertAid<br />

Premium was able to amplify 0.1pg of RNA. Amplification of<br />

the human PPP1CA gene was performed on varying amounts<br />

of Jurkat cell total RNA. First strand cDNA was generated with<br />

the Maxima ® Kit and amplified with the Maxima ® Probe /ROX<br />

qPCR Master Mix (2X) using the TaqMan ® assay specific for<br />

PPP1CA. Reactions were performed on an ABI 7500. Reaction<br />

efficiency was 105%, slope-3.29, R 2 =0.999. (Figure 4)<br />

Maxima ® First Strand cDNA Synthesis Kit for RT-qPCR<br />

• Sensitive and reproducible cDNA synthesis from<br />

a wide range of starting RNA amounts (0.01 pg - 5 μg).<br />

• Increased synthesis rate – complete cDNA synthesis<br />

in 15 minutes.<br />

• Increased reaction temperature – up to 60°C.<br />

• Convenient format – premixed solutions for use in RTqPCR.<br />

• First strand cDNA was generated from 100 ng-1 pg<br />

of total Jurkat cell RNA with the Maxima ® First Strand<br />

cDNA Synthesis Kit for RT-qPCR (#K1641) in 16 replicate<br />

reactions.<br />

Components:<br />

• Maxima ® Enzyme Mix<br />

• 5X Reaction Mix<br />

• Water, nuclease-free<br />

• Amplification of the E.coli 23S RNA gene was performed<br />

on 10-fold serial dilutions of total RNA (30 ng to 3 fg). First<br />

strand cDNA was generated using the Maxima ® First Strand<br />

cDNA Synthesis Kit for RT-qPCR (red) and QuantiTect<br />

reverse transcription kit from Qiagen (green). cDNA was<br />

amplified using Maxima ® SYBR Green/ROX qPCR Master<br />

Mix (#K0221) on the ABI 7500 Real-Time PCR instrument.<br />

Figure 4 - High sensitivity and capacity in two step RT-qPCR<br />

<strong>Thermo</strong> Scientific offer a number of Reverse Transcriptase<br />

solutions including stand alone enzymes and kits. Enzymes<br />

available in standalone format include RevertAid Premium<br />

Reverse Transcriptase and Maxima ® Reverse Transcriptase.<br />

Kits are also available for use in RT-qPCR (Maxima ® First Strand<br />

cDNA Synthesis Kit for RT-qPCR) and RT-PCR (RevertAid<br />

Premium First Strand cDNA Synthesis Kit).<br />

Greater yields and higher sensitivity<br />

Amplification of the E.coli 23S<br />

RNA gene was performed on<br />

10-fold serial dilutions of total RNA<br />

(30 ng to 3 fg). First strand cDNA<br />

was generated using the Maxima ®<br />

First Strand cDNA Synthesis Kit for<br />

RT-qPCR (red) and QuantiTect<br />

reverse transcription kit from<br />

Qiagen (green). cDNA was<br />

amplified using Maxima ® SYBR<br />

Green/ROX qPCR Master Mix<br />

(#K0221) on the ABI 7500<br />

Real-Time PCR instrument.<br />

27


Pioneering<br />

in PCR Technology<br />

References:<br />

1. Kleppe K, Ohtsuka E, Kleppe<br />

R, Molineux I and Khorana HG<br />

“Studies on polynucleotides.<br />

XCVI. Repair replications<br />

of short synthetic DNA’s as<br />

catalyzed by DNA polymerases.”<br />

J. Mol. Biol. 56: 341-361<br />

(1971).<br />

2. Saiki RK et al.<br />

“Enzymatic amplifcation of<br />

ß-globin genomic sequences<br />

and restriction site analysis for<br />

diagnosis of sickle cell anemia.”<br />

Science 230: 1350-54 (1985).<br />

3. Mullis KB “Process for<br />

amplifying nucleic acid<br />

sequences” U.S. Patent<br />

4,683,202.<br />

4. Saiki RK, Gelfand DH,<br />

Stoffel S, Scharf SJ, Higuchi<br />

R, Horn GT, Mullis KB. and<br />

Erlich HA “Primer-directed<br />

enzymatic amplifcation of<br />

DNA with a thermostable<br />

DNA polymerase.” Science,<br />

239:487-91 (1988).<br />

5. Guyer RL, and Koshland<br />

DE “The Molecule of the Year.”<br />

Science 246:1543-1546<br />

(1989).<br />

PCR is a biochemical process for amplifying large quantities<br />

of particular DNA sequences from relatively small amounts of<br />

starting material. The basic concepts involved in PCR were<br />

described as early as 1971 1 , but it was not until 1985 that they<br />

were reduced to practice by Kary Mullis and patented at Cetus<br />

Corporation 2,3 . PCR revolutionised molecular biology almost<br />

overnight, and Kary Mullis was awarded the Nobel Prize in<br />

1993 – within a decade of inventing the method.<br />

Extending the possibilities<br />

Since its invention, improvements in PCR have largely<br />

depended on advancements in enzymology. The first great<br />

breakthrough in PCR technology was the replacement of<br />

1953<br />

The discovery of the<br />

DNA double helix<br />

structure<br />

PCR<br />

Throughout<br />

the Ages<br />

1967<br />

5’ exonuclease activity allowed this polymerase to proof<br />

read as it extended the new DNA strand, and correct errors<br />

as it progressed. This resulted in an overall increase in<br />

fidelity several fold, and as a bonus also greatly improved<br />

the ability of this enzyme to successfully amplify long PCR<br />

products (Taq polymerase, typically being stalled by its own<br />

misincorporations, is quite poor at amplifying targets larger<br />

than a few kilobases).<br />

The specificity of PCR can be adversely affected by nonspecific<br />

binding of the primers to themselves or to non-target<br />

DNA, which can occur during the time between reaction set<br />

up and the first denaturation step. To avoid this, “hot start”<br />

1950 1955 1960 1965 1970 1975 1980 1985<br />

1970<br />

Thomas Brock<br />

reports on the<br />

isolation of the<br />

extremophilic<br />

bacterium<br />

<strong>Thermo</strong>philis<br />

aquaticus<br />

Kleppe and<br />

co-workers first<br />

describe a method<br />

using an enzymatic<br />

assay to replicate a<br />

short DNA template<br />

with primers in vitro<br />

1976<br />

Taq polymerase is<br />

isolated (a<br />

thermostable DNA<br />

polymerase named<br />

after the thermophilic<br />

bacterium Thermus<br />

aquaticus)<br />

1977<br />

1982<br />

Frederik Sanger and colleagues<br />

introduce the “dideoxy”<br />

chain-termination method for<br />

sequencing DNA (also known as<br />

‘Sanger sequencing’). It utilises DNA<br />

polymerase, nucleotide precursors<br />

and one oligonucleotide primer<br />

The first cited use of microarray technology<br />

(Augenlicht LH, Kobrin D. “Cloning and<br />

screening of sequences expressed in a<br />

mouse colon tumour.” Cancer Research 42<br />

(3): 1088-1093, 1982.<br />

http://cancerres.aacrjournals.org/content/4<br />

2/3/1088.long<br />

1983<br />

1985<br />

PCR technique is<br />

described in an<br />

article published<br />

in Science (Saiki<br />

et al)<br />

Working for Cetus, Kary Mullis<br />

discovers that using two<br />

oligonucleotides instead of one - on<br />

opposite strands - enables DNA to<br />

be synthesised from a single,<br />

specific, location in the genome.<br />

Technique for PCR was created.<br />

Escherichia coli (E. coli) DNA polymerase (or its derivative<br />

Klenow Fragment) with a thermostable polymerase from<br />

Thermus aquaticus (Taq) 4 . This polymerase could withstand<br />

the high temperatures required during the denaturation and<br />

extension steps of PCR, and thus fresh enzyme was not<br />

required during each cycle.<br />

Taq polymerase was commercialised in the late 1980s,<br />

spurring a boom in PCR and ultimately becoming Science<br />

magazine’s first “molecule of the year” in 1989 5 . Although a<br />

vast improvement over using E. coli Pol I, Taq polymerase<br />

still had some serious drawbacks. Limited thermostability at<br />

temperatures above 94°C reduced the enzymes effectiveness<br />

when targeting regions with high GC-content or particularly<br />

strong secondary structure, plus a lack of proofreading ability<br />

rendered Taq rather error prone, thus complicating cloning and<br />

sequencing projects that utilised PCR. The race was now on to<br />

develop even better polymerases for PCR.<br />

The pursuit of the advanced polymerase<br />

The first thermophilic DNA polymerase exhibiting improved<br />

fidelity was characterised by the founders of Finnzymes<br />

Oy (now part of <strong>Thermo</strong> <strong>Fisher</strong> Scientific) in 1991 6 . A 3’ to<br />

techniques were developed by several groups independently<br />

in the late 1980s. The first manual hot start methods involved<br />

simply heating up the PCR reaction to 95°C and then cooling<br />

to 60-70°C before adding the polymerase; this method<br />

was effective but tedious and carried the risk of crosscontamination<br />

of samples when the hot tubes were opened.<br />

By 1991, new products were developed such as waxes that<br />

could be used to physically separate the enzyme from the<br />

PCR mix until initial denaturation.<br />

As so often in the history of PCR, however, the enduring<br />

technological advancement involved the polymerase. By<br />

adding a specific antibody (or similar molecule such as an<br />

aptamer or an Affbody ® ) that inactivates the polymerase, the<br />

enzyme itself can be bestowed with hot start functionality,<br />

as demonstrated by two groups independently in 1994 7,8 .<br />

By 1997, a chemical modification of DNA polymerase was<br />

demonstrated to produce similar results, although the<br />

enzyme reactivated over the course of the PCR, and thus<br />

behaved differently to the antibody-based versions.<br />

Mimicking nature – Phusion proteins<br />

Further improving on naturally occurring DNA polymerases<br />

28


1989<br />

Science<br />

Magazine<br />

names Taq<br />

polymerase it‘s<br />

first “Molecule<br />

of the year.“<br />

1989<br />

Kary Mullis<br />

received the<br />

Nobel Prize<br />

The first real-time<br />

PCR instrument is<br />

described<br />

1995<br />

The first complete<br />

genome of a<br />

free-living organism is<br />

sequenced by Venter<br />

and colleagues<br />

(Haemophilus<br />

influenzae)<br />

2000<br />

Lynx Therapeutics<br />

published and markets<br />

“MPSS” - a parallelised,<br />

adapter/ligation-mediated,<br />

bead-based sequencing<br />

technology, launching “next<br />

generation” sequencing<br />

2007<br />

The fist<br />

complete<br />

human<br />

genome is<br />

sequenced by<br />

Levy et al.<br />

2010<br />

Gibson et al. create the<br />

first bacterial cell<br />

controlled by a<br />

chemically synthesised<br />

genome (using Phusion<br />

High Fidelity DNA<br />

Polymerase)<br />

1990 1995 2000 2005 2010<br />

1988<br />

Patent for Taq DNA polymerase<br />

is filed by Mullis et al.<br />

The first automated PCR cycler<br />

is introduced to the market by<br />

Perkin Elmer and Cetus (joint<br />

venture)<br />

1991<br />

The first<br />

high-fedelity<br />

DNA<br />

polymerase is<br />

characterised<br />

by Mattila et al.<br />

required a technological leap. The breakthrough innovation<br />

was to introduce fusion protein technology into DNA<br />

polymerases 9 . The first of these next generation polymerases<br />

was Phusion High Fidelity DNA polymerase. Phusion is an<br />

engineered enzyme combining an Archaebacterial polymerase<br />

possessing strong proofreading ability with a thermostable<br />

double-stranded DNA binding protein. This binding protein<br />

increases affinity of the polymerase for double-stranded<br />

DNA, functionally mimicking the processivity factors that are<br />

normally found in the complex DNA replisomes of live cells.<br />

The high processivity and robustness of Phusion make the<br />

enzyme extremely resistant to PCR inhibitors and enable<br />

additional applications not feasible with other more standard<br />

polymerases. These properties were exploited with the<br />

introduction of Direct PCR kits that allow PCR to be performed<br />

directly from a wide range of starting material, without the<br />

need for sample preparation or DNA isolation. Kits have been<br />

developed allowing PCR to be performed directly from blood<br />

as well as a variety of plant, animal and human tissues.<br />

1994<br />

Antibody<br />

based hot<br />

start<br />

technology<br />

described<br />

1996<br />

Evolution of enzymes in vitro<br />

The idea of using rational design criteria to improve enzyme<br />

performance is limited by our knowledge of polymerase<br />

Genome of the first eukaryotic<br />

organism, Saccharomyces<br />

cerevisiae, is sequenced<br />

Two commercial real-time<br />

PCR instruments are launched<br />

to market<br />

2003<br />

Phusion High-Fedelity<br />

DNA Polymerase, the<br />

first PCR enzyme<br />

based on fusion<br />

protein technology, is<br />

launched by<br />

Finnzymes Oy<br />

fine structure and function. However, Fermentas’ patented<br />

technology for in vitro protein evolution (2009), enabled<br />

the introduction and selection of favourable mutations into<br />

polymerases and transcriptases. The Maxima and RevertAid<br />

Premium Reverse Transcriptases are examples of enzymes<br />

that were derived by in vitro evolution of M-MuLV RT. The<br />

resulting enzymes show a 50x increase in processivity<br />

compared to their wild-type counterparts, and are highly<br />

thermostable at temperatures up to 65ºC. Additionally, similar<br />

to Phusion DNA polymerases, Maxima and RevertAid Premium<br />

RTs demonstrate increased resistance to reaction inhibitors<br />

such as guanidine or formamide.<br />

A fusion of synergistic technologies<br />

The acquisitions of Finnzymes and Fermentas by <strong>Thermo</strong><br />

<strong>Fisher</strong> Scientific have brought together a wide variety of<br />

technologies, and these synergies will allow for exciting<br />

new product development in the field of PCR and RT-PCR.<br />

Finnzymes’ advanced PCR enzymes together with the trusted<br />

Fermentas PCR and reverse transcriptase enzymes derived<br />

from in vitro evolution make a powerful combination that will<br />

help scientists in molecular biology continue to make new<br />

discoveries and drive innovation.<br />

2009<br />

The MIQE guidelines<br />

(Minimum Information for<br />

Publication of<br />

Quantitative Real-Time<br />

PCR Experiments) are<br />

published by Bustin et al<br />

6. Mattila P, Korpela J,<br />

Tenkanen T and Pitkänen K<br />

“Fidelity of DNA synthesis by<br />

the <strong>Thermo</strong>coccus litoralis DNA<br />

polymerase - an extremely heat<br />

stable enzyme with proofreading<br />

activity” Nucleic Acids Res.<br />

19: 4967-4973 (1991).<br />

7. Sharkey DJ, Scalice ER,<br />

Christy Jr KG, Atwood SM<br />

and Daiss JL. “Antibodies as<br />

thermolabile switches: high<br />

temperature triggering for the<br />

Polymerase Chain Reaction.”<br />

Nature Biotechnology 12:<br />

506 - 509 (1994).<br />

8. Kellogg DE, Rybalkin I,<br />

Chen S, Mukhamedova N,<br />

Vlasik T, Siebert PD, Chenchik<br />

A. “TaqStart antibody: hot start<br />

PCR facilitated by a neutralizing<br />

monoclonal antibody directed<br />

against Taq DNA polymerase.”<br />

BioTechniques 16 (6):1134-<br />

1137 (1994).<br />

9. Wang Y, Prosen DE, Mei L.<br />

Sullivan JC, Finney M, and Vander<br />

Horn PB “A novel strategy<br />

to engineer DNA polymerases<br />

for enhanced processivity<br />

and improved performance<br />

in vitro.” Nucleic Acids Res.<br />

32:1197-1207 (2004).<br />

29


DNA Quantification in<br />

with <strong>Thermo</strong> Scientific<br />

SP&A Application Laboratory // <strong>Thermo</strong> <strong>Fisher</strong> Scientific Vantaa, Finland<br />

Introduction<br />

UV photometry is a common way to quantify nucleic acids in<br />

a sample. Both DNA and RNA absorb UV light very efficiently,<br />

making it possible to detect and quantify their concentrations.<br />

Typical applications for this include, for example, the<br />

quantification of template prior to sequencing or PCR.<br />

The photometric method is based on Lambert-Beer’s<br />

equation and it utilises the fact that the nitrogenous bases<br />

in nucleotides have an absorption maximum at about 260<br />

nm. The average extinction coefficient for double-stranded<br />

DNA is 0.020 (μg/ml) -1 cm -1 . This means that 1.0 Abs at 260<br />

nm corresponds to a concentration of 50 μg/ml for doublestranded<br />

DNA. Thus the amount of DNA can be calculated<br />

by using the formula: DNA concentration (μg/ml) = Abs260 x<br />

50 μg/ml.<br />

Unlike the nucleic acids, proteins have a UV absorption<br />

maximum at 280 nm, mostly due to the tryptophan residues.<br />

Therefore, the Abs260/Abs280 ratio gives an estimate of<br />

the protein contamination of the sample. For a good quality<br />

sample, the value should be between 1.8 and 2.0. A value<br />

smaller than 1.8 indicates the presence of proteins and a<br />

value higher than 2.0 indicates probable contamination,<br />

such as phenols. Another common parameter used to<br />

describe the quality of DNA is the 260-to-230 nm ratio. It is<br />

used to estimate chemical contamination, such as phenols,<br />

carbohydrates or a high salt concentration. The ideal 260/230<br />

nm ratio is around 2.<br />

The amount of sample available for the analysis is quite often<br />

very low, a tool such as the μDrop Plate and the <strong>Thermo</strong><br />

Scientific NanoDrop, enable these measurements at a<br />

microliter scale.This article will discuss the use of the μDrop<br />

Plate as an option for low-volume quantitation with a<br />

Microplate UV-VIS spectrophotometer.<br />

Figure 1. μDrop Plate.<br />

A low sample volume<br />

measurement area<br />

with 16 measurement<br />

locations on the left<br />

and a cuvette location<br />

for a 10 mm cuvette<br />

on the right.<br />

The μDrop Plate’s low-volume measurement area consists of<br />

two quartz slides, the top clear quartz slide and the bottom<br />

partially Teflon-coated quartz slide. The bottom slide contains<br />

16 sample positions, arranged in a 2 x 8 matrix, onto which<br />

samples can be pipetted. The cuvette slot of the plate is used<br />

to perform photometric measurements with standard cuvettes.<br />

Compared to a normal cuvette; the pathlength of the μDrop<br />

Plate low-volume area is short; 10 mm vs. 0.5 mm. By<br />

decreasing the pathlength the sample volume can also be<br />

decreased. A sample volume down to 2 μl can be used with<br />

the μDrop Plate.<br />

A shorter pathlength also increases the requirements of the<br />

photometer used as it reduces the measured absorbance.<br />

It is possible to measure DNA concentrations from a few<br />

nanograms to thousands of nanograms per microliter with a<br />

μDrop Plate and a photometer with high precision and a wide<br />

linear range.<br />

Any photometric measurement device; cuvette, microplate<br />

or μDrop Plate, always has certain background absorption.<br />

Therefore, blank subtraction is always necessary when<br />

photometric quantification of the sample concentrations<br />

is performed.<br />

• <strong>Thermo</strong> Scientific μDrop Plate, N12391<br />

• <strong>Thermo</strong> Scientific Multiskan GO microplate and cuvette<br />

spectrophotometer, <strong>Thermo</strong> Scientific, 51119300<br />

• <strong>Thermo</strong> Scientific Varioskan Flash multimode reader,<br />

<strong>Thermo</strong> Scientific, 5250030<br />

• Semi-micro cuvette, Hellma, 104-10-40-QS<br />

• Herring Sperm DNA (solution was serially diluted<br />

in a ratio of 1:2 into TE buffer to provide 7 sample<br />

concentrations)<br />

The concentrations of the samples were measured with the<br />

μDrop Plate low-volume area, the cuvette position and the<br />

Multiskan GO cuvette port. The concentrations measured<br />

with the Multiskan GO cuvette port were used as references<br />

in all the calculations. The sample volume used was 4 μl in<br />

all low-volume area tests. For the cuvette measurements,<br />

the DNA samples were diluted 1:20 to keep the absorbance<br />

values within the linear measurement range.<br />

A blank measurement was made with 16 replicates before<br />

the concentration measurements. The average of the blank<br />

samples was subtracted from all unknown absorbances.<br />

30


Micro-litre Volumes<br />

µDrop Plate<br />

Terminology<br />

The sensitivity of the assay is determined according to IUPAC with two different parameters: Limit of Detection (LOD) and Limit<br />

of Quantification (LOQ)<br />

LOD is the lowest amount of analyte that can be separated from the background. It is calculated based on the calibration<br />

curve slope vs. the blank + 3 x SD of the blank. LOD means, that this amount of analyte can be detected with statistical<br />

significance, but not necessarily quantified as an exact value.<br />

LOQ is the lowest amount of the analyte at which quantification is possible with statistical relevance. It is defined as the<br />

quantitative detection limit as Cld = ks/m, where k is 10, s is the standard deviation of instrument readings taken on blank, and<br />

m is the slope of a plot of instrument response vs. concentration, as calculated by linear regression.<br />

In practice, LOD is the limiting value in qualitative assays where a simple yes/no answer to the question “Is there any analyte<br />

in my sample” is required and LOQ is the limiting value that the user can measure as the concentration of the analyte in<br />

quantitative assays.<br />

Results<br />

The results obtained with the µDrop Plate low-volume<br />

area and the cuvettes were compared to the results of the<br />

Multiskan GO cuvette port results.<br />

Sensitivity<br />

The limit of detection and quantification were calculated as<br />

described above. An average value of several µDrop Plate<br />

measurements are shown in Table 1.<br />

Limit of detection<br />

(LOD)<br />

Limit of<br />

quantification<br />

(LOQ)<br />

Multiskan GO<br />

Varioskan Flash<br />

1.7 µg/ml 2.2 µg/ml<br />

5.6 µg/ml 7.2 µg/ml<br />

Table 1. LOD and LOQ values of the DNA<br />

quantification assay with the uDrop Plate and<br />

the Multiskan GO or Varioskan Flash.<br />

Detection range<br />

The measured concentrations with the Drop Plate low-volume<br />

area in the Multiskan GO was compared to the values of the<br />

Multiskan GO cuvette port (Figure 2). The system showed<br />

excellent linearity up to the highest sample concentration: ><br />

2000 μg/ml.<br />

µDrop Plate (µg/ml)<br />

2500<br />

2000<br />

1500<br />

1000<br />

500<br />

Multiskan GO<br />

y=1.006x - 4.0148<br />

R 2 =1<br />

These values are well within the precision specifications of<br />

the Multiskan GO and Varioskan Flash, 9µg/ml.<br />

0<br />

0 500 1000 1500 2000 2500<br />

Multiskan GO cuvette (µg/ml)<br />

Summary<br />

» It is possible to measure nucleic acid concentrations from micrograms to even milligrams per<br />

milliliter with the µDrop Plate.<br />

» The fixed pathlength of the plate enables direct calculation of the nucleic acid concentrations.<br />

» The results of the low-volume area correlate very well with the cuvette measurement results.<br />

References<br />

1) Mocak et al.,<br />

Pure and Applied<br />

Chemistry, Vol. 69 No.2,<br />

pp 297–328, 1997.<br />

31


High-content gene expression<br />

analysis with TRAC<br />

Rautio Jari PlexPress, Helsinki, Finland, Sini Suomalainen <strong>Thermo</strong> <strong>Fisher</strong> Scientific, Vantaa, Finland<br />

Introduction<br />

Years of intensive global gene expression studies have<br />

yielded an abundance of genome-wide expression data<br />

enabling identification of gene expression signatures for<br />

diverse biological states such as disease states, patient<br />

responses or toxicological responses.<br />

The subsequent need is to analyse focused gene sets from<br />

large data samples efficiently and cost efficiently for research,<br />

drug screening and diagnostic purposes.<br />

TRAC (Transcript analysis with aid of affinity capture) is a<br />

novel hybridisation and bead-based assay enabling multiplex<br />

mRNA target detection simultaneously from large sample<br />

numbers.<br />

TRAC Technology<br />

The TRAC method enables rapid quantification of focused<br />

gene sets from a large number of samples. Cellular material<br />

is collected and lysed. Transcript levels are measured directly<br />

from the resulting cell lysate without the need for RNA<br />

purification or qRT-PCR amplification.<br />

Each gene of interest is recognised by a specific<br />

complementary fluorophore-labeled probe. A pool of probes<br />

with different lengths or types of labels is added to each<br />

sample with a hybridisation buffer. Biotin-oligo-dT is used to<br />

capture targets from their polyA tails.<br />

Hybridisation of probes and transcripts takes place in the<br />

solution, a faster and more reproducible process than<br />

hybridisation in which one partner is bound to a surface.<br />

User Benefits<br />

High information value:<br />

• Multiplexing allows for a thorough study of<br />

gene function<br />

• Observing the dynamics of gene expression gives greater<br />

insight into gene functions<br />

• Powerful research data improve decision making to reduce<br />

project time and lower project costs<br />

Simplicity:<br />

• Direct use of cell lysates; no RNA extraction<br />

• No cDNA conversion required<br />

• Simple assembly of new, custom-made gene sets<br />

• Easy implementation; experiments are simple to set up<br />

Speed:<br />

• 96-Well plate format enables automated, high throughput<br />

sample processing<br />

• Rapid assay protocol with little hands-on time<br />

Accuracy:<br />

• Liquid carryover between processing steps is minimised to<br />

reduce the risk of contamination<br />

• Degradation of RNA is eliminated<br />

• Control genes are incorporated into the multiplexed<br />

mixture, results are normalised<br />

Accuracy:<br />

• Intra and inter-assay CVs


WORKFLOW CELLS TO DATA<br />

1<br />

Sample Preparation<br />

• Remove media<br />

• Add lysis buffer<br />

2<br />

Hybridisation<br />

• Add samples and hybridisation<br />

buffer on 96-well plate<br />

• Hybridise at +60 °C, 30-90 min.<br />

3<br />

4<br />

5<br />

Dispense on<br />

King<strong>Fisher</strong> Plates<br />

Capture, Wash<br />

and Elute with<br />

King<strong>Fisher</strong><br />

Instrument<br />

Fragment Analysis<br />

• 5x Wash buffer (WB) plates<br />

• 1x Elution buffer (EB)<br />

• 1x Bead plate<br />

• Samples after hybridisation<br />

• Insert plates and proceed<br />

according to TRAC-specific<br />

protocol<br />

• Insert plates to a DNA sequencer<br />

and proceed according to<br />

TRAC-specific fragment protocol<br />

6<br />

Parsing<br />

• Analyse the raw data from<br />

the DNA analyser using<br />

TRACParser Software<br />

TRAC application examples<br />

siRNA knockdown validation<br />

Objective<br />

TRAC assay was used to optimise siRNA delivery conditions<br />

(transfection and exposure times and cell amounts) and to<br />

evaluate androgen receptor (AR) siRNA knockdown efficiency.<br />

The TRAC results were compared to luciferase reporter<br />

gene assay.<br />

Results and conclusions<br />

TRAC and luciferase assays showed consistent variations in<br />

the efficiency of target silencing by different siRNA products.<br />

Also, some non-targeting control siRNAs occurred at high<br />

concentrations to decreased levels of AR transcript. In<br />

addition to AR transcription, TRAC enabled simultaneous<br />

detection of 14 other genes related to AR response,<br />

interferon response and cell viability.<br />

Cancer marker screening in<br />

cell cultures<br />

Objective<br />

TRAC assay with the King<strong>Fisher</strong> instrument was used to<br />

screen expression signatures of 20 cancer-related gene<br />

markers in colon cancer cell lines cultured on 96-well<br />

plates. The gene markers were related to cell adhesion,<br />

angiogenesis and plasminogen activation. The assay was set<br />

up for use with chemical-based gene expression screening<br />

of cell cultures. Expression profiles of four different cell lines<br />

(COLO, HT-29, CaCo2 and DLD) were compared with gene<br />

markers after treatment with a drug candidate.<br />

Results and conclusions<br />

The expression signatures could be detected directly from<br />

10 – 100 x 10 3 cells grown on 96-well plates. The dynamics<br />

of gene expression for the analysed set could not have been<br />

observed at a single point in time. Reproducibility was good<br />

with CVs below 12% for the system.<br />

Literature<br />

1. Rautio JJ et al (2008)<br />

TRAC in high-content<br />

gene expression analysis:<br />

Applications in Microbial<br />

populatiion studies, process<br />

biotechnology and<br />

biomedical research.<br />

Expert Rev. Mol. Diagn.<br />

8, 379-385.<br />

33


Separation of<br />

influenza virus<br />

Technical Note:<br />

Influenza viruses are spherical in shape with diameters ranging between 80 &<br />

120nm. They consist of an enveloped RNA & are classified in the Orthomyxoviridae<br />

family. There are three kinds of influenza viruses depending on<br />

serotype: type A, type B, and type C. However antigenic changes<br />

within these subtypes results in the extreme diversity of viral strains<br />

and makes an annual reformulation of the influenza vaccine<br />

necessary 1 . In recent years highly virulent avian and swine flu<br />

viruses have been raising serious concerns about flu pandemics.<br />

34


Procedures<br />

This brief describes a protocol for the separation of influenza virus using the <strong>Thermo</strong> Scientific S50-A fixed angle rotor and <strong>Thermo</strong><br />

Scientific Sorvall Micro-Ultracentrifuges. This rotor provides the largest capacity in the <strong>Thermo</strong> Scientific micro-ultracentrifuge family<br />

and allows sample processing up to 180 mL. All centrifugation steps were performed using the <strong>Thermo</strong> Scientific S50-A fixed-angle<br />

rotor with 25 mL open-top polycarbonate (PC) thick-walled tubes (PN 75000610, actual volume = 19.8 mL) and the <strong>Thermo</strong> Scientific<br />

Sorvall MTX 150 Micro-Ultracentrifuge; a <strong>Thermo</strong> Scientific Sorvall MX Micro-Ultracentrifuge may alternatively be used.<br />

Separation Procedures<br />

1 Remove host derived coarse foreign substances from<br />

infected allantoic fluid or infected cell culture medium by<br />

performing centrifugation with the following parameters:<br />

6,000 rpm for 20 minutes. Note: Depending on volume,<br />

this step can be completed in a floor model superspeed<br />

centrifuge, such as the <strong>Thermo</strong> Scientific Sorvall RC 6<br />

Plus, or a general purpose centrifuge.<br />

2 Pour the supernatant into PC thick-walled tubes. Note:<br />

PC thick-walled tubes must be filled to a minimum of<br />

50% capacity.<br />

3 Perform centrifugation using the S50-A fixed-angle rotor<br />

with the following parameters: 32,000 rpm (~85,800 x g)<br />

for 45 minutes at 4°C, Acc.9, Dec. 7.<br />

4 Remove the supernatant and add 1.5 mL of Veronal<br />

buffer solution including 3 mM CaCl 2<br />

to the sediment, to<br />

minimise the formation of viral-containing clumps.<br />

5 Resuspend by pipetting and leave overnight at 4 °C.<br />

6 Layer the concentrated virus fluid over 17 mL of a 10 to<br />

40% (w/v) sucrose continuous density gradient solution in<br />

each PC thick-walled tube.<br />

7 Perform centrifugation using the S50-A fixed-angle rotor<br />

with the following parameters: 32,000 rpm (~85,800 x g)<br />

for 45 minutes, at 4 °C, Acc.9, Dec. 7.<br />

8 A white layer is formed slightly above the centre of the<br />

tube. The virus layer can be observed, in a dark room, by<br />

exposing light to the tube. Collect the minimum amount of<br />

virus layer (up to ~ 2 mL).<br />

9 Dilute the fractionated virus fluid with 1.5 times buffer<br />

solution (fluid volume after dilution: ~ 3 mL). Layer the<br />

diluted virus fluid over 15 mL of a 30 to 60% (w/v) sucrose<br />

continuous density gradient solution in each PC thickwalled<br />

tube.<br />

10 Perform centrifugation using the S50-A fixed-angle rotor<br />

with the following parameters : 32,000rpm (~85,800 x g)<br />

for 45 minutes at 4 °C, Acc.9, Dec. 7.<br />

11 Collect the formed virus layer & dilute it with 2.5 times or<br />

more buffer solution to bring the total volume to 17–18 mL.<br />

12 Perform centrifugation using the S50-A fixed-angle rotor<br />

with the following parameters: 32,000 rpm (~85,800 x g)<br />

for 60 minutes at 4 °C, Acc. 9, Dec. 7. Add buffer solution<br />

to the sediment and resuspend.<br />

Conclusion<br />

The protocol referenced in this brief allows viral particles to be<br />

isolated efficiently through a sucrose gradients using the S50-A<br />

fixed-angle rotor and Sorvall ® MTX 150 or Sorvall MX Series<br />

Micro-Ultracentrifuge. The S50-A rotor allows for the isolation<br />

of virus particles in a large volume that was previously reserved<br />

for standard ultracentrifuges and rotors.<br />

References<br />

1. Biosafety in Microbiological<br />

and Biomedical Laboratories,<br />

5th Edition (2007). US Dept. of<br />

Health and Human Services,<br />

Public Health Service, Centres<br />

for Disease Control and Prevention<br />

and National Institutes<br />

of Health.<br />

2. Hitachi Koki Co. (2008).<br />

Separation of Influenza<br />

Viruses Using Fixed Angle Rotor<br />

Designed for Tabletop Micro<br />

Ultracentrifuge<br />

35


high throughput<br />

RNA isolation<br />

from bovine endometrial epithelial cells<br />

Abstract<br />

Optimal reproductive performance is essential for the survival of each species and the economic basis<br />

for dairy enterprises. Implantation and maintenance of pregnancy are critical phases after fertilisation and<br />

can be affected by several factors including the expression of cytokines, hormones, interleukins and<br />

prostaglandins. In order to investigate the mRNA expression pattern of fertilisation relevant<br />

factors under multiple experimental conditions, it is necessary to adopt an efficient and<br />

reproducible system for high throughput RNA isolation. mRNA quantification via realtime<br />

RT-PCR includes potential sources of error, which can lead to incorrect results or<br />

to variation in the expression level. A detection of the RNA integrity (RIN) helps to<br />

standardise the RNA quantification and reflect the RNA quality. RNA purification<br />

from hundreds of samples often represents a bottleneck in sample analysis.<br />

The use of an automated, easy-to-handle method is therefore highly desirable<br />

to increase productivity and reproducibility. In this study we report the<br />

successful application of the Stratec InviMag Universal RNA Mini Kit on the<br />

King<strong>Fisher</strong> Flex 96 from <strong>Thermo</strong> Scientific in order to investigate the expression<br />

of different factors playing a key role for fertilisation.<br />

Methods<br />

Oviducts were collected from the slaughterhouse as previously described (1) .<br />

Due to the fact that experiments with animals should be avoided or replaced<br />

by in vitro approaches, establishment of cell cultures models were introduced.<br />

For the RNA isolation, cells after first passage were cultured to confluence in 6 well<br />

plates and exposed to media with different nutrient compositions for 12 h, 24 h or 48 h,<br />

respectively. Highly pure total RNA was isolated using the InviMag Universal RNA Mini Kit on<br />

the King<strong>Fisher</strong> Flex 96. The King<strong>Fisher</strong> technology is based on magnetic rods transferring<br />

particles through the various purification phases. The lysis step is done outside the workstation<br />

and the carrier bound DNA is removed by centrifugation. The RNA containing supernatant is transferred<br />

to the King<strong>Fisher</strong> deep well plate and ethanol as well as magnetic particles are added followed<br />

by automated RNA purification on the King<strong>Fisher</strong> platform. The preparation time for 96 samples in<br />

parallel takes approx. 1 h – 1 h 30 min.<br />

36


Results<br />

These kit performances allow the isolation of highly pure<br />

RNA without any DNase digestion step.<br />

1 The high purity of the isolated RNA is shown in a RIN<br />

Factor of about 10.0.<br />

2 The yield of total RNA was between 10 - 20 µg per well<br />

depending on the animal and growing rate.<br />

3 The ratio A260: A280 is in average 2.05 (standard<br />

deviation +/-0.2%) measured by a NanoDrop photometer.<br />

Summary<br />

The use of the InviMag Universal RNA Mini Kit on the King<strong>Fisher</strong><br />

Flex 96 provides the following advantages:<br />

a High quality total RNA recovery without DNA digestion<br />

• RIN factor in average of 9.9<br />

• Ratio A260: A280 2.05<br />

b Fast isolation of RNA from 96 samples in approx. 60 min<br />

c Convenient handling<br />

d Error prevention for high throughput analysis<br />

All samples are processed on the same plate, at the<br />

same time and using standardised highly reproducible<br />

procedures (automated system)<br />

Conclusion<br />

In conclusion the use of the InviMag Universal RNA Mini Kit on<br />

the King<strong>Fisher</strong> Flex 96 for the analysis of the expression pattern<br />

of fertilisation relevant factors underlines the RNA integrity, the<br />

efficiency, reproducibility and convenience of this tool for high<br />

throughput RNA isolation.<br />

37


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Innovations-Magazine-<strong>Issue</strong>1.indd 1<br />

1<br />

8/11/2010 9:27:14 AM<br />

1<br />

Q&A<br />

Q. Should I choose probe or SYBR Green chemistry for my<br />

qPCR experiments?<br />

A. When you are optimising a new experiment and you need to test<br />

primer pairs, it is usually best to start with SYBR Green. SYBR Green<br />

enables you to find the most specific primers without the need to<br />

design and purchase several probes. SYBR Green allows melting<br />

curve analysis, which can reveal undesirable primer-dimers or other<br />

nonspecific PCR products.<br />

Probe chemistries are better when you require more specificity in<br />

detection (e.g. discrimination between closely related species). Probes<br />

are also needed for multiplex reactions that allow you to detect multiple<br />

targets at once.<br />

Q. Why is the amplicon length so limited in qPCR?<br />

A. The amplification efficiency tends to decrease as the amplicon length<br />

increases. Short amplicons are more likely to be completely denatured<br />

during the denaturing step, making the annealing of primer and probes<br />

more efficient. Also, shorter amplicons are copied more rapidly during<br />

the extension step.<br />

Pass it on If you have enjoyed reading<br />

Bio-Innovation please pass it on to a colleague.<br />

They may enjoy reading the magazine and could<br />

learn something that could move their research<br />

forward.<br />

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Bio-Innovation and have received this copy from<br />

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register for your own copy email:<br />

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Publish in Bio-Innovation If you’ve used our<br />

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would benefit from the information generated,<br />

why not share it with the wider scientific<br />

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Tell us what you think. We would like to get<br />

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so that we can improve it and increase your<br />

reading enjoyment. Send us your comments or<br />

suggestions for improvement to<br />

Bio-Innovation@thermofisher.com<br />

Q. Why is my amplification efficiency over 100%? Why is my<br />

standard curve not a straight line?<br />

A. PCR efficiency depends on the method used to determine it. If the<br />

efficiency is calculated based on a standard curve, the slope of the curve<br />

may be skewed at one end or the other by the late detection of a large<br />

concentration of product due to inhibitors, or by the early detection of<br />

non-specific PCR products or primer-dimers. The skewed slope directly<br />

affects the calculated efficiency. An efficiency over 100% can also be the<br />

result of an inaccurate standard dilution series. This can be avoided by<br />

careful use of calibrated pipettors when preparing standard dilutions.<br />

Bio<br />

Bio<br />

Q. Why do I get several peaks on my SYBR Green melting curve<br />

even though the amplification efficiency is good?<br />

A. There can be several reasons for abnormal melting curve data.<br />

Non-specific amplification does not necessarily have an impact on the<br />

amplification efficiency and even if it does, the effect can be difficult to<br />

see. Additional peaks may also form as a result of primers binding to a<br />

closely related gene. It is important to check primers for specificity using<br />

a BLAST search.<br />

Q. How many reference genes do I need?<br />

A. In relative quantification, reference genes are used to normalise<br />

against variation in sample quality and quantity. The number of reference<br />

genes needed depends on the assay, the accuracy needed and the<br />

references chosen. Several reference gene candidates should be tested<br />

to find the most suitable combination. There are many software tools<br />

available for validation of reference genes.<br />

Bio<br />

02<br />

DNA Detective<br />

Recent Developments in<br />

qPCR detection chemistries<br />

Life’s better in Colour<br />

<strong>Thermo</strong> <strong>Fisher</strong> Scientific<br />

Celular Imaging Competition<br />

Groundbreaking research<br />

The discovery and verification of<br />

cardiovascular & stroke biomarkers<br />

Bio-I novation-Magazine-I sue(2).in d 1 6/01/20 1 4:01:43 PM<br />

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