RNA Guide: Purifying RNA and mRNA - Promega

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RNA Guide: Purifying RNA and mRNA - Promega

Purifying RNA and mRNA

Purification Requirements

The purity and integrity of RNA is critical to the success

of any RNA-based analysis. Purification methods must

protect RNA during and after purification.

To successfully isolate intact RNA by any procedure,

the methods must:

1. Disrupt cells or tissue.

2. Denature the nucleic acid:protein complexes.

3. Inactivate endogenous ribonucleases.

4. Purify the RNA from contaminating DNA and

protein.

Do you need total RNA or poly(A)+ RNA for your

application? For most applications, total RNA is

sufficient, but in some

cases the added

sensitivity of poly(A)+

RNA is beneficial. This

is especially true if the

RNA is intended for

cDNA library

construction or

detection of very

rare messages.

RNasin ®

Ribonuclease Inhibitor

protects purified

RNA in downstream

applications.

See page 3

Look for these symbols to find the system

right for your application.

Animal Tissue

Cultured Cells

Blood

Promega has a variety of solutions for your RNA

purification needs. All systems produce high-quality

RNA ready for use from a variety of starting materials.

The PureYield RNA Midiprep System uses a novel

procedure for removal of genomic DNA from the RNA

prep without DNase treatment. The RNA is ideally suited

for any application influenced by the presence of

genomic DNA, such as quantitative, real-time RT-PCR.

The SV, SV 96 and MagneSil ® Total RNA Isolation

Systems include DNase for in-process genomic DNA

removal. Both the SV 96 and MagneSil RNA Systems

can be automated for high-throughput purification. The

RNAgents ® System is a total scalable solutions-based

system that uses organic extraction to produce high

quality RNA.

Plant Tissue

5584MA

Bacteria

Yeast

Please note, in the flow charts that follow, Promega systems

are listed based on the protocols are provided in the Technical

Literature sent with each system. Other applications with other

source materials may exist.

Please contact Promega Technical Services if you have

questions: techserv@promega.com

www.promega.com • techserv@promega.com 7


Purifying RNA and mRNA

Automated

Animal

Tissue

Manual

Unique product!

Novel clearing agent

removes all detectable

genomic DNA.

No DNase

treatment needed!

MagneSil ® Total RNA

mini-Isolation System

SV 96 Total RNA

Isolation System

PureYield RNA

Midiprep System

Can process up to

2mg of tissue lysate

in 200µl of lysis

buffer in the

96-well format

DNase included to

greatly reduce

gDNA carry-over.

Scalable solution-based

reagent that can

process up to 1g of

tissue in a single prep

SV Total RNA

Isolation System

RNAgents ® Total

RNA Isolation system

PolyATtract ® System

1000

5585MA

Go from tissue to

poly(A)+ RNA

directly.

The SV Total RNA Isolation System has been cited for total

RNA isolation from a wide variety of mammalian tissues

including heart, lung, kidney, brain, eye, colon, small intestine,

muscle, cochlea, stomach, placenta and thymus. Some more

exotic tissues include:

snake venom sacs & glands

insect larvae

whole water flea

zebrafish gut

carp eggs

frog liver & gonads

chicken heart, leg & brain

marine polychaetes

For more information, check the online citations

database at: www.promega.com/citations or contact

Technical Services at: techserv@promega.com

8

Promega RNA Analysis Notebook


Purifying RNA and mRNA

Cultured

Cells

Unique product!

Novel clearing agent

removes all detectable

genomic DNA.

No DNase

treatment needed!

Automated

Manual

MagneSil ® Total RNA

mini-Isolation System

SV 96 Total RNA

Isolation System

PureYield RNA

Midiprep System

SV Total RNA

Isolation System

Can process: ~10 5

cells in 96-well

format; s10 3 cells in

384-well format

Precipitation of RNA Samples

DNase included to

greatly reduce

gDNA carry-over.

Precipitation of RNA is commonly used for purification

or concentration purposes. To ensure maximum

recovery when precipitating small quantities (


Purifying RNA and mRNA

Automated

Blood

Manual

RNA yield

from blood

varies with

white cell count

MagneSil ® Total RNA

mini-Isolation System

PureYield RNA

Midiprep System

SV Total RNA

Isolation System

Unique product!

Novel clearing agent

removes all detectable

genomic DNA.

No DNase

treatment needed!

RNAgents ® Total

RNA Isolation System

Scalable solution-based

reagent that can

process up to 1gm of

tissue in a single prep

5587MA

DNase included to

greatly reduce

gDNA carry-over.

Can process 20µl

whole blood in

96-well format;

5µl whole blood in

384-well format

Protocols require

removal of red blood

cells with SV Red

Cell Lysis Solution

prior to purification

In addition to RNA isolation from mammalian

blood, the SV Total RNA Isolation System has

been used on related non-mammalian sources,

including marine invertebrate hemolymph and

fish red blood cells.

10

Promega RNA Analysis Notebook


Purifying RNA and mRNA

Plant

Tissue

Manual

PureYield RNA

Midiprep System

SV Total RNA

Isolation System

RNAgents ® Total

RNA Isolation System

5588MA

Unique product!

Novel clearing agent

removes all detectable

genomic DNA.

No DNase

treatment needed!

DNase included to

greatly reduce

gDNA carry-over.

Scalable solution-based

reagent that can

process up to 1gm of

tissue in a single prep

The SV Total RNA Isolation System has been cited for total RNA purification from tissues of the following:

Arabidopsis thaliana

Beta vulgaris

Brassica napus

Cichorium intybus

Hordeum vulgare

Lotus japonica

Lycospersicon esculentum

Manihot exculenta

Medicago truncatula

Nicotiana tabacum

Pisum sativum

Prunus cerasifera

Trifolium repens

Vigna sp.

Vitis vinifera

Tissues used for isolation include root, stem, leaves, seedlings and berries. For more information, check the online

citations database at: www.promega.com/citations or contact Technical Services at: techserv@promega.com

www.promega.com • techserv@promega.com 11


Purifying RNA and mRNA

Protocols require

lysozyme and/or

lysostaphin

Bacteria

Manual

Protocols for

Gram Positive &

Gram Negative

bacteria

SV Total RNA

Isolation System

DNase included to

greatly reduce

gDNA carry-over.

PureYield RNA

Midiprep System

Unique product!

Novel clearing agent

removes all detectable

genomic DNA.

No DNase

treatment needed!

5589MA

The SV Total RNA Isolation System has been

cited for total RNA purification from many

bacteria. Some examples are:

Alteromonas

Bacillus

Chlamydia

Escherichia

Klebsiella

Lactobacillus

Microcystis

Mycobacterium

Neisseria

Orchrobactrum

Photorhabdus

Pseudomonas

Rhizobium

Rhodopseudomonas

Salmonella

Simkania

Streptococcus

Streptomyces

Synechocystis

Vibrio

Yersinia

For more information, check the online

citations database at:

www.promega.com/citations/

or contact Technical Services at:

techserv@promega.com

Protocols require

lyticase or

zymolase

Manual

Yeast &

Fungi

The SV Total RNA Isolation System has been

cited for total RNA purification from:

Saccharomyces

cerevisiae

Blumeria grominis

Bierkandera sp.

Gigaspora margarita

Trichophyton rubrum

SV Total RNA

Isolation System

PureYield RNA

Midiprep System

5590MA

12

Promega RNA Analysis Notebook


Purifying RNA and mRNA

Scalable Total RNA Isolation

Promega’s original solution-based system, the

RNAgents ® Total RNA Isolation System is based on the

classic method described by Chomczynski and Sacchi

(1). We improved the method by developing a novel

Denaturation Solution that reduces DNA carryover.

This scalable system can be used with mammalian

tissue, cultured cells and plant tissue and can be

adapted to any sample size.

10

8

RNAgents ® Total RNA

Isolation System

Protocol available at:

www.promega.com/tbs/tb087/

tb087.html

Cat.# Z5110

Citations available at:

www.promega.com/citations/

1 2 3 4 5 6

RNA Yield (mg)

6

4

– 28S

– 18S

Formaldehyde gel electrophoresis of total RNA. Five micrograms of total RNA

isolated with RNAgents ® System from HeLa cells (lane 1), mouse intestine (lane 2), mouse

spleen (lane 3), mouse lung (lane 4), mouse kidney (lane 5) and mouse liver (lane 6).

0349TA11_5A

2

Add Sodium

Acetate to

prepared lysate.

0

0 0.2 0.4 0.6 0.8 1.0

Tissue (g)

Purification of total RNA from mouse liver with RNAgents ® Total RNA

Isolation System.

1270MA11_5B

Table 1. Yields and A 260 /A 280 Ratios of Total RNA purified

with the RNAgents ® System.

Source Yield of Total RNA A 260 /A 280

HeLa Cells 1.6mg/10 8 cells 1.85

Human WBC 1.3mg/10 8 cells 1.72

Mouse Intestine 2.3mg/g tissue 1.75

Mouse Spleen 8.3mg/g tissue 1.67

Mouse Lung 1.9mg/g tissue 1.75

Mouse Liver 6.6mg/g tissue 1.99

Mouse Kidney 3.1mg/g tissue 1.70

RNAgents ® Denaturing

Solution is available separately

and can be easily combined

with your reagents providing

an economical choice for

RNA isolation.

Add Phenol:

Chloroform:

Isoamyl Alcohol

to tube, mix

and chill.

Transfer mixture

and centrifuge.

Transfer top aqueous

phase to new tube.

Add Isopropanol.

Incubate at –20°C.

Centrifuge, then

discard supernatant.

For RT-PCR go to "RNA Wash"

The RNAgents ® Procedure.

Add Denaturing

Solution. Vortex.

Add Isopropanol.

Centrifuge, then

discard supernatant.

Add ice-cold

75% ethanol.

Centrifuge, then

discard supernatant.

Air-dry the RNA

pellet. Dissolve in

water or TE buffer.

Store.

3513MA09_2B

www.promega.com • techserv@promega.com 13


Purifying RNA and mRNA

Small-scale Single-prep

Total RNA Isolation

The single-prep SV Total RNA Isolation System offers

speed and convenience. The column-based method

can isolate total RNA from up to 60mg of mammalian

tissue. Denaturation and inactivation of RNases are

accomplished without the use of phenol. Promega was

the first to offer a system that eliminates residual DNA

by performing DNase digestion directly on the column

membrane, greatly reducing DNA carryover.

SV Total RNA Isolation System

Protocol available at:

www.promega.com/tbs/tm048/

tm048.html

Cat.# Z3100

Citations available at:

www.promega.com/citations/

Max. Amount Avg.

Sample Type Processed Yield

Mouse Liver 30mg 131µg

Mouse Kidney 20mg 44µg

Mouse Spleen 15mg 79µg

Mouse Brain 60mg 39µg

Mouse Muscle 30mg 22µg

Rat Pancreas 30mg 100µg

Rat Heart 60mg 16µg

Rat Lung 60mg 36µg

Tomato Leaf 30mg 5µg

E. coli 10 9 cells 36µg

S. cerevisiae 4 × 10 7 cells 19µg

Spin

Spin for one minute.

Wash with RNA

Wash Solution.

DNase Treatment.

Add DNase

Stop Solution.

Spin for one minute.

Wash 2X with

RNA Wash Solution.

Elute RNA into

Elution Tube.

DNase

treatment gives

better results

+DNase

Homogenize sample (tissues,

cultured cells or white blood cells)

in RNA Lysis Buffer.

Transfer 175µl to a fresh tube. Add

RNA Dilution Buffer; mix and centrifuge.

Transfer the cleared lysate to a fresh tube;

add 95% Ethanol and mix.

Transfer to Spin Column Assembly.

Total time: 60-70 minutes.

–DNase

DNase

included

Vacuum

Remove lysate

by vacuum.

Wash with RNA

Wash Solution.

DNase Treatment.

Add DNase Stop Solution.

Wash 2X with RNA

Wash Solution.

Remove Spin Basket and place

in collection tube. Centrifuge.

Place Spin Basket in Elution Tube.

–1.2Kb

–400bp

1766MB04_1A

Taken from Technical Manual #TM048

3548TA09_1A

RT-PCR amplification from total RNA purified from mouse liver lysate with or

without DNase treatment. Amplification primers used for RT-PCR were designed to

amplify both mRNA and DNA in the same reaction. Amplification products specific for the

mRNA are 400bp, and amplification products for the gene are 1.2kb. Both the SV and SV

96 Total RNA Isolation Systems include DNase for on-membrane DNase treatment to

remove genomic DNA below detectable levels.

14

Promega RNA Analysis Notebook


Purifying RNA and mRNA

Mid-scale, Single-prep Total

RNA Isolation

As standard molecular biology applications become more

sensitive, it is increasingly important for RNA isolations

to be free of contaminating genomic DNA. The

PureYield RNA Midiprep System uses a novel combination

of reagents, membrane, and protocol to achieve

pure RNA with undetectable genomic DNA contamination.

The isolation is completed without the use of a

DNase treatment, organic solvents, protease digestions,

or alcohol precipitations. The eluted RNA is ready for

sensitive downstream applications such as quantitative

RT-PCR, RT-PCR, and microarray analysis.

Amplification Curves

No detectable

gDNA by qPCR

1. Add isopropanol to the

cleared lysate.

2. Mix and transfer to

PureYield Binding Column.

1. Prepare lysate.

2. Add RNA Dilution Buffer. Mix.

3. Add Clearing Agent. Mix and vortex.

4. Incubate at 70°C for 5 minutes.

5. Cool for 5 minutes.

1. Transfer mixture to PureYield

Clearing Column in collection tube.

2. Centrifuge to clear the lysate.

No DNase

treatment

required

RFU

-1.1E5 -1.0E5 -9.0E5 -8.0E5 -7.0E5 -6.0E5 -5.0E5 -4.0E5 -3.0E5 -2.0E5 -1.0E5 0

DNA Standards

Competitor I

No-Template Control

PureYield

RNA Midiprep

5 10 15 20 25 30 35 40

Cycle

RNA purified with the PureYield RNA Midiprep System has no detectable

genomic DNA contamination. RNA was purified from 1 × 10 8 HEK293T using the

PureYield system and competitor “I’s” solution-based purification method. 100ng of

purified total RNA was analyzed using the Plexor qPCR System to determine the

quantity of gDNA using primers specific for the human thyroid peroxidase gene

(TPOX). Human Genomic DNA (Cat.# G3051) in quantites of 10 4 , 10 3 , 10 2 and

10 1 copies was used as a standard.

PureYield RNA Midiprep

System

Protocol available at:

www.promega.com/tbs/tm279/tm

279.html

Cat. # Z3740 and Z3741

5360TA

Centrifuge (Spin)

1. Centrifuge.

2. Wash 2X.

3. Centrifuge

10 minutes

to dry.

Vacuum (Vac)

1. Apply vacuum.

2. Wash 2X.

3. Dry 3 minutes.

1. Transfer PureYield Binding

Column to 50ml collection tube.

2. Add Nuclease-Free Water

and incubate 2 minutes.

3. Centrifuge 3 minutes to elute

pure RNA.

Schematic representation of the PureYield Total RNA Isolation System.

Average Yields of Total RNA Isolated from Tissues and Cells.

Sample

Sample Amount / Maxium Average Yield Average Average

Type Sample Capacity per Prep (µg) 1 A 260 /A 230 A 260 /A 280

Rat Tissues

Liver 300mg 990.7 1.8 1.9

Lung 300mg 193.9 2.0 2.1

Kidney 200mg 329.1 2.3 2.1

Spleen 150mg 430.9 2.3 2.1

Brain 300mg 305.5 2.3 2.1

Heart 300mg 255.3 2.2 2.1

Muscle 300mg 115.1 2.1 2.1

Bacteria

E. coli 1 × 10 10 cells 872.7 2.5 2.1

Plant Tissue

Canola 300mg 87.8 1.5 2.1

Cell Lines

HEK 293T 5 × 10 7 cells 453.3 2.1 1.9

HeLa 5 × 10 7 cells 329.2 1.8 2.0

Blood 20ml (10ml/tube) ~10* * *

1. The values represent means of results achieved at Promega. Yields will depend on the metabolic state of

the sample, culture conditions, harvesting conditions and sample preparations. The average total RNA yield

shown is from a 1ml elution.

* Varies by white cell count. A white cell count of ~5 × 10 6 cells/ml yields ~10µg of total RNA.

www.promega.com • techserv@promega.com 15

5124MB


Purifying RNA and mRNA

96-Well Total RNA Isolation

As your RNA isolation needs grow, Promega has the

tools to increase your RNA isolation throughput. All the

advantages of the single-prep SV Total RNA Isolation

System are included in a 96-well format of the SV 96

Total RNA Isolation System. The system uses a vacuum

manifold to allow the isolation of RNA from an entire

plate of 96 samples as efficiently as possible. Isolations

can be scaled up for benchtop purification or for use

with liquid-handlers. Use the Vac-Man ® 96 Vacuum

Manifold for benchtop and automated methods unless

your robotic system requires a specific manifold.

SV 96 Total RNA Isolation

System

Protocol available at:

www.promega.com/tbs/tb294/

tb294.html

Cat.# Z3500, Z3505

Automated protocols available.

Apply sample lysate

Binding Plate

Bind RNA.

Wash.

DNase treat.

DNase

included

Wash.

Elute total RNA.

3446CA06_1A

Elution Plate

Highly pure total RNA.

2651MB03_1A

16

Comparison of SV and SV 96 Total RNA Isolation

Yields

Total Yield (µg)

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

SV 96

If it works in SV,

it’ll work in SV 96!

0

1 × 10 5 Cells

Vacuum

Spin

Standard SV Total

RNA preps

3806MA08_2A

β-actin –

Go directly from

eluted RNA to

RT-PCR!

RT-PCR from a Decreasing Number of SH-SY5Y

Human Neuroblastoma Cells

SH-SY5Y/SV96

50k

25k

12.5k

6,250

3,125

1,562

781

391

195

98

49

24

RNA was isolated from individual wells containing the indicated number of

cells using the SV 96 Total RNA Isolation System. Four microliters of each eluate

were amplified with the Access RT-PCR System (Cat.# A1250) and β-Actin Primer Pair

(Cat.# G5740).

Promega RNA Analysis Notebook

3344TA03_1A


Purifying RNA and mRNA

µg

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

RNA isolated with the

SV 96 Total RNA System can

go directly into quantitative

RT-PCR reactions.

HeLa NIH 3T3 CHO

SV96

3467MA06_1A

∆Rn

5.0

4.0

3.0

2.0

1.0

0

0 5 10 15 20 25 30 35 40

Cycle

Number of HeLa Cells

50,000 12,500

25,000 6,250

3,125

1,563

4068TA03_3B

Ratio A 260 /A 280

2.5

2.0

1.5

1.0

0.5

0

HeLa NIH 3T3 CHO

SV96

Ribosomal RNA Sizes

Species rRNA Size (kb)

Human 18S 1.9

28S 5.0

Mouse 18S 1.9

28S 4.7

Drosophila 18S 2.2

28S 2.8

Tobacco 16S 1.5

18S 1.9

23S 2.9

25S 3.7

Yeast 18S 2.0

26S 3.8

E. coli 16S 1.5

23S 2.9

3468MA06_1A

Real-time quantitative RT-PCR analysis of lamin A/C message. Total RNA

isolated from indicated number of HeLa cells and eluted in 100µl of Nuclease-Free Water.

Twenty microliters were used in a 100µl RT reaction and 5µl transferred to a 50µl

quantitative, real-time PCR reaction. Further details in Brisco, P. and Hooper, K. (2003)

Quantitative, real-time RT-PCR expression using the SV 96 Total RNA Isolation System.

Promega Notes 84, 23–26.

Storage of purified RNA

RNA should be stored in a dedicated container at

–70°C or below. To avoid multiple freeze-thaw cycles

and contamination, dispense the purified RNA into

convenient volumes. For long-term storage or storage

of critical samples, precipitate RNA aliquots by adding

1/10th volume of 3M sodium acetate and 2 volumes

of ethanol. Store the precipitated RNA under ethanol

at –70°C or below.

SV 96 Total RNA

works on plant and

mammalian tissues.

Tomato Corn Tobacco Barley Wheat Alfalfa Arabid. Soy Rice

M L S L S L S L S L S L S M L L L

3509TA07_1B

Isolation of total RNA from either leaf (L) or stem (S) tissues of various plant

species. Bands indicate 28S, 18S and chloroplast rRNAs. Twenty microliters of each

elution were loaded on a 1% gel stained with ethidium bromide. Protocol detailed in

Grunst, T. (2001) High-throughput isolation with the SV 96 Total RNA Isolation System.

Promega Notes 79, 29–32.

www.promega.com • techserv@promega.com

17


Purifying RNA and mRNA

96-Well or 384-Well Total

RNA Isolation

The MagneSil ® Total RNA mini-Isolation System

provides a high-throughput 96- or 384-well format for

fast, simple preparation of intact, purified total RNA

from small amounts of cultured cells, tissue, or fresh

whole blood samples. The system is designed to

perform four plates worth of purification in a 96- or

384-well plate format. Total RNA isolation is achieved

without the need for vacuum filtration, centrifugation or

precipitation. The procedure is specifically geared for

automated liquid handlers.

Add sample lysate to

MagneSil ® RNA PMPs. Mix.

Capture PMPs.

Discard supernatant.

Wash PMPs.

Incubate PMPs with

DNase 10 minutes.

Add DNase Stop Solution.

Elute RNA from a

96-well purification

in as little as 15µl

Capture PMPs.

Discard supernatant.

Wash PMPs.

Concentration (ng/µl)

30

20

10

0

10 15 20 25 30 35 40 45 50

Elute RNA.

Schematic diagram of the MagneSil ® Total RNA mini-Isolation System

protocol.

4316MB11_3A

Elution Volume (µl)

Yield (µg)

0.6

0.4

0.2

0.0

10 15 20 25 30 35 40 45 50

Elution Volume (µl)

Flexibility in elution volume to control sample concentration. Total RNA isolated

from 2 × 10 4 HeLa cells/well using MagneSil Total RNA mini-Isolation System in a 96-well

format. Titration of elution volume shown. Total RNA concentration and yield calculated by

measurement of isolated total RNA with Molecular Probes RiboGreen ® assay.

4377MA11_3A

Maximum starting material for MagneSil ® Total RNA

mini-isolation System protocols:

Sample 96-well 384-well

Tissue Lysate

Not

(in 100µl lysis buffer) 2mg recommended

Cultured Cells 10 5 10 3

Whole Blood 20µl 5µl

The mobile stationary phase

of magnetic particles allows

elution in smaller volumes than

membrane-based RNA

purification systems

18

Promega RNA Analysis Notebook


Purifying RNA and mRNA

Ready for quantitative,

real-time RT-PCR

A.

∆Rn

Ct

10 2

10 1

10 0

10 –1

10 –2

40

30

20

10 3

10 4 10 2

10 5 10

10 –3 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Cycle

GAPDH

negative control

Automated Methods for:

• Beckman Coulter Biomek ® 2000

• Beckman Coulter Biomek ® FX

• Tecan Te-MO

• Xiril X100

Only the Biomek ® FX and Te-MO can apply the 384-well

method. Some instruments may need extra hardware to

automate this system For more information go to:

www.promega.com/automethods/

If you don’t have one of the instruments listed, you may

be able to adapt the system to your instrument. Our

reagent manuals include information such as how much

of each reagent are required at each step to assist with

the adaptation.

10

B.

∆Rn

10 0

10 –1

0

1

10

100

1,000

Cell Number

10,000

100,000

1E+06

10 1 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

10 5 10 4 10 3 10 2

MagneSil ® Total RNA

mini-isolation System Protocol

available at:

www.promega.com/tbs/tb328/

tb328.html

Cat.# Z3351

10 –2

10

Automation and magnetic stands

required for use.

10 –3

Cycle

40

c-myc

30

Ct

20

10

0

1

10

100

1,000

Cell Number

10,000

100,000

1E+06

4378TA11_3A

Real-time RT-PCR analysis of purified RNA. 20µl aliquots of total RNA isolated

from a 10X dilution series of HeLa cells seeded in a 96-well plate were used as template

for reverse transcription (100µl reaction). Panel A: 5µl aliquots (n=3) of the RT reaction

were used for PCR of GAPDH target. Panel B: 5µl aliquots (n=1) of the RT reaction were

used for PCR of a c-Myc target. GAPDH signal (abundant mRNA) and c-Myc (rare mRNA)

could be detected in as few as 10 HeLa cells.

www.promega.com • techserv@promega.com 19


Purifying RNA and mRNA

Poly(A)+ mRNA Isolation

Many applications are improved by isolating poly(A)+

RNA either directly from the eukaryotic source material

or from total RNA previously isolated from the source

material. Some methods use oligo(dT) immobilized on a

support such as cellulose or sepharose. These methods

are prone to ribosomal RNA carryover. Promega’s

PolyATtract ® Systems use solution hybridization of

Poly(A)+ RNA to a biotinylated oligo(dT) followed by

capture with a streptavidin-coated paramagnetic

particle. The rapid hybridization and magnetic

separation greatly improve the speed, efficiency and

quality of mRNA isolations.

The PolyATtract ® mRNA Isolation Systems can be used

if you are starting with total RNA. The kits are designed

to purify Poly(A)+ RNA from 1–5mg or 0.1–1mg of total

RNA. Reactions are scalable to meet your needs.

To save time, isolate poly(A)+ RNA directly from starting

material using the PolyATtract ® System 1000. The

system uses a guanidine solution to disrupt the tissue

and inhibit RNases, followed by the PolyATtract ®

methodology for the isolation of Poly(A)+ RNA.

Promega has published applications for mammalian

and plant tissues.

From Total RNA...

PolyATtract ® mRNA Isolation System

Protocols available at:

www.promega.com/tbs/tm021/tm021.html

Cat.# Z5200, Z5210, Z5300, Z5310

Citations available at:

www.promega.com/citations/

total RNA containing

mRNA fraction

5′ m7G AAAAAAA

3′

Hybridize with

biotin-oligo(dT).

5′ m7G AAAAAAA 3′

3′ TTTTTTT-B

5′

Add streptavidin PMPs.

PMP

5′ m7G AAAAAAA

3′

TTTTTTT-B

Magnetize.

Wash and elute.

5′ B-TTTTTTT

3′

PMP

5′ m7G AAAAAAA

3′

TTTTTTT-B

PMP

MAGNET

Direct Isolation...

PolyATtract ® System 1000

Protocols available at:

www.promega.com/tbs/tm228/tm228.html

Cat.# Z5420, Z5400

See citations at:

www.promega.com/citations/

5′ m7G AAAAAAA

3′

(aqueous)

Solution-based

hybridization to oligo(dT)

is more efficient than

hybridization to an

immobilized oligo(dT)

TTTTTTT-B

(solid)

PMP

MAGNET

µg total RNA µg mRNA

12 6 3 1.5 2 1 0.5 M

0369MA03_1A

20

Prokaryotic mRNA enrichment

Bacterial mRNA does not have the poly(A) tail

characteristic of eukaryotic mRNA. Other tactics

must be employed if you wish to get at the bacterial

mRNA. Su and Sordillo (1998) used a probe directed

to bacterial rRNA to remove the rRNA from a total

bacterial RNA prep to enrich the sample for the

bacterial mRNA.

Su, C. and Sordillo, L.M. (1998) A simple method to

enrich mRNA from total prokaryotic RNA. Mol.

Biotechnol. 10, 83–85.

Northern blot analysis

comparing total RNA and

poly(A)+ RNA isolated

from mouse liver. The

Northern blot was probed for

low abundance α-1-proteinase

inhibitor message. Poly(A)+

RNA was isolated directly from

the tissue with the

PolyATtract ® System 1000.

Promega RNA Analysis Notebook

0417TA06_2A


Purifying RNA and mRNA

Downstream Applications

Expression Profiling

See chapter 5, Analyzing RNA with Microarrays.

See chapter 3, Quantifying RNA with qRT-PCR.

RT-PCR

See chapter 4, Amplifying RNA with RT-PCR.

Primer Extension Analysis

Primer extension is used to quantitate and determine

the location of the 5′-end of specific RNAs. With this

technique, a radiolabeled DNA primer complementary to

the RNA being studied is hybridized to the target and

extended using the enzymatic properties of reverse

transcriptase. The DNA primer is designed to anneal to

sequences near the 5′-end of the RNA target, and the

extension reaction terminates when the reverse

transcriptase reaches the extreme 5′-end of the RNA.

The length of the cDNA product accurately defines the

distance between the 5′-end of the radiolabeled primer

and the 5′-end of the RNA (transcriptional start site).

The quantity of cDNA produced is proportional to the

amount of target RNA.

Primer Extension System

Protocol available at:

www.promega.com/tbs/

tb113/tb113.html

Cat.# E3030

Need information on other

applications?

Contact Promega Technical Services

techserv@promega.com

Nuclease Protection

The nuclease protection assay is a sensitive technique

used for the detection and quantitation of target RNA

sequences and related RNAs (6). A radiolabeled DNA or

RNA probe is allowed to hybridize to target RNA in

solution. After hybridization, the remaining singlestranded

probe is removed from the reaction by

incubation with either a ribonuclease, if the probe was

RNA (RNase protection), or S1 nuclease, if the probe

was DNA (S1 assay). Reaction products are resolved by

polyacrylamide gel electrophoresis to quantitate the

amount of protected probe. The technique is extremely

sensitive, doesn’t require transfer of RNA to a solid

support, and multiple RNA species can be probed in a

single reaction. See the chapter called “Making RNA in

vitro” for information about making RNA probes.

Promega also offers

RNase ONE

Ribonuclease, a

highly efficient

ribonuclease that

cleaves after each

base in RNA so you

won’t have to use

mixtures like RNase A

and RNase T1.

RNase ONE Ribonuclease

Cat.# M4261

Citations available at:

www.promega.com/citations/

Northern Blotting

Northern blotting is the classic technique used to

examine the expression profile of mRNA following a

specific treatment. Northern analysis has given way to

techniques like RT-PCR and microarray analysis. For a

complete method for Northern blotting, see the

Expression Analysis chapter of the Protocols and

Applications Guide, available online at:

www.promega.com/paguide.

Protocols & Applications

Guide at:

www.promega.com/paguide

www.promega.com • techserv@promega.com 21


Purifying RNA and mRNA

Total RNA Purification Products Size Cat.#

SV Total RNA Isolation System (d) * 10 preps Z3101

50 preps Z3100

PureYield RNA Midiprep System (c,e) 10 preps Z3740

50 preps Z3741

SV 96 Total RNA Isolation System* 1 × 96 Z3500

5 × 96 Z3505

RNAgents ® Total RNA Isolation System* Scalable Z5110

MagneSil ® Total RNA mini-Isolation System (f) 4 plate Z3351

Items Available Separately

SV RNA Lysis Buffer* 50ml Z3051

SV RNA Red Cell Lysis Buffer* 200ml Z3141

Used to lyse red blood cells prior to isolation of RNA from the nucleated white blood cells.

Vac-Man ® Laboratory Vacuum Manifold 1 each A7231

For use with the SV Total RNA System in a vacuum format.

Twenty samples can be processed at once.

Vacuum Adapters 20 each A1331

Required for use with the Vac-Man ® Laboratory Vacuum Manifold and the

SV Total RNA Isolation System in a vacuum format.

Vac-Man ® 96 Vacuum Manifold 1 each A2291

Required for use of the SV 96 Total RNA Isolation System.

RNAgents ® Denaturing Solution 120ml Z5651

mRNA Purification Products Size Cat.#

PolyATtract ® mRNA Isolation System I* 3 isolations Z5200

1–5mg total RNA; Magnetic Stand included

PolyATtract ® mRNA Isolation System II* 3 isolations Z5210

Refill system for Z5200; no magnetic stand

PolyATtract ® mRNA Isolation System III* 15 isolations Z5300

0.1–1mg total RNA; Magnetic Stand included

PolyATtract ® mRNA Isolation System IV* 15 isolations Z5310

Refill system for Z5300; no magnetic stand

PolyATtract ® System 1000 with Magnetic Stand* 3 isolations Z5420

PolyATtract ® System 1000 without Magnetic Stand* 3 isolations Z5400

*For Laboratory Use.

References

1. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by

acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem.

162, 156–159.

2. Marcus, L. et al. (1996) PolyATtract ® Systems for mRNA purification. Promega

Notes 60, 14–16.

3. Murillo, I. et al. (1995) Isoaltion of total RNA and mRNA from plant tissues.

Promega Notes, 2–5.

4. Rhodes, R. and Kephart, D. (1996) Automated Robotic Isolation of Poly(A)+

mRNA Using PolyATtract ® mRNA Isolation Reagent. Promega Notes 75, 10–13.

5. Barlow, J.J. et al. (1963) A simple method for quantitative isolation of

undegraded high molecular weight ribonucleic acid. Biochem. Biophys. Res.

Comm. 13, 61–66.

6. Ausubel, F.M. et al. (2000) Current Protocols in Molecular Biology, Vol. 2, John

Wiley & Sons, New York, 4.6–4.7.

22

Promega RNA Analysis Notebook

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