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