mrna detection
TRANSCRIPT
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2 Promega Notes 77
By Donna Leippe, Ph.D., Ken Lewis, Ph.D., John Shannonhouse, M.S., John Shultz, Ph.D., and Scott Weatherbee*, Ph.D.
Promega Corporation
The Poly(A) mRNA Detection System
A BSTRACT
The Poly(A) mRNA Detection System is a luciferase-based
system designed for the specific and sensitive detection of
polyadenylated messenger RNA. Detection is based on a series
of enzymatic reactions resulting in a light signal proportional
to the amount of poly(A) mRNA in the sample. The system is
specific; minimal signal is generated from ribosomal or trans-
fer RNAs. The system is also sensitive and can detect picogram
levels of mRNA, levels that cannot be detected by conventional
methods such as spectrophotometry. This also minimizes the
amount of valuable sample committed to measurement pur-
poses. This article demonstrates the linearity, specificity and
sensitivity of this new system.
INTRODUCTION
The measurement of nucleic acids is important for many tech-
niques in molecular biology. As techniques both generate and
require less sample, the need for sensitive measurements increases.
We are developing technologies for sensitive and specific nucleic
acid detection to address such needs. The Poly(A) mRNA
Detection System(a) is suitable for the detection of picogram to
nanogram levels of polyadenylated mRNA. It possesses superior
sensitivity compared to spectrophotometry or gel electrophoresis.The enhanced sensitivity also reduces the fraction of sample used
for measurement purposes.
Poly(A) mRNA is specifically targeted in this assay through
hybridization to oligo(dT) primers. In contrast to spectrophotometry,
this system distinguishes mRNA from other RNA species such as
ribosomal and transfer RNA, which may be present in RNA prepa-
rations. Gel electrophoresis can distinguish between these species
but has limited usefulness for determining the concentration of a
heterogeneous population of mRNA molecules, which might
appear as multiple bands of 500–3,000 bases in length (1,2). Since
The Poly(A) mRNA Detection System is a
luciferase-based system designed for the
specific and sensitive detection of
polyadenylated messenger RNA.
the system is specific for poly(A) mRNA, the value obtained may
vary greatly from the amount estimated by absorbance or dye
intercalation techniques, which measure all RNA species.
The Poly(A) mRNA Detection System is ideal for the detection of mRNA when small amounts or dilute samples are the starting
material. A knowledge of mRNA concentration and quality is use-
ful prior to RT-PCR(b) or cDNA construction. It can also facilitate
the normalization of mRNA levels prior to generating cDNA
probes for gene arrays.
HOW THE POLY (A) MRNA DETECTION S YSTEM WORKS
The technology of the Poly(A) mRNA Detection System is based
on a series of enzymatic reactions to detect double-stranded nucleic
acids. The technology takes advantage of the pyrophosphorylation
activity of polymerases (3), which carries out the depolymerization
of double-stranded nucleic acids. By changing the polymerase and
reaction conditions, different nucleic acid moieties can be specifi-
cally detected (4). For the Poly(A) mRNA Detection System,
Klenow Fragment, Exonuclease Minus, is used to detect poly(A)
RNA in the presence of excess rRNA and tRNA. The pyrophos-
phorylation activity of DNA Polymerase I (Klenow Fragment)
allows the depolymerization of dsDNA (3) and the DNA strand of
DNA:RNA hybrids. Hybridization of an anchored oligo(dT) to
poly(A) RNA followed by pyrophosphorylation allows the specific
detection of the poly(A) fraction of RNA. The oligo(dT) primers
are 18mers that have a single deoxyguanosine, deoxycytidine or
deoxyadenosine base at the 3´-end. This base at the 3´-end serves to
*Current Address: Memorial Sloan-Kettering Cancer Center, New York, NY
Figure 1. Detection of nucleic acids using the Poly(A) mRNA Detection System. VTTTTT represents the 18-base
oligonucleotide, where V = A, G or C.
mRNA
DNA Polymerase I(Klenow), Exonuclease Minus
Pyrophosphate ADP dNDP
dNTP
NDPK
ATPLuciferase-produced
light
AAAAAAAAAAVTTTTT
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direct and “anchor” the oligonucleotide to the start of the poly(A)
tail. Klenow Fragment, Exonuclease Minus, lacks 5´→3´ and
3´→5´ exonuclease activities and will only pyrophosphorylate
DNA strands that are perfectly matched at their 3´-termini.
The hybridization step is followed by an incubation during which
two coupled enzymatic reactions (pyrophosphorylation and
transphosphorylation) are performed (Figure 1). The first reaction
is the pyrophosphorylation of the anchored oligo(dT) hybridized tothe poly(A) segments of mRNA. The products of this pyrophos-
phorylation reaction are free nucleoside diphosphates (dNTPs),
where the amount of free nucleotide produced is dependent upon
the amount of hybridized anchored oligo(dT). In the second reac-
tion, the terminal phosphate from the dNTP is transferred to ADP
to form ATP. This reaction is catalyzed by a nucleoside diphos-
phate kinase (NDPK, 5). The net result of these two reactions is
the production of an amount of ATP proportional to the number
of poly(A) tails in the sample. The ATP is a substrate for the
luciferase enzyme (6) and is measured in a third reaction with the
highly sensitive Luciferase/Luciferin Reagent. Figure 2 shows a
schematic of the overall protocol. The main interfering substances
are ATP and other nucleoside and deoxynucleoside triphosphates,
ATPases, nucleases and dsDNA.
As mentioned above, the DNA Polymerase I (Klenow) Fragment,
Exonuclease Minus, will pyrophosphorylate only hybridized, prop-
erly anchored oligo(dT), not free oligo(dT) or bound oligonu-
cleotides with mismatched 3´-termini. This is demonstrated by the
data in Table 1. A synthetic 1.2kb Kanamycin mRNA, which has a
cytosine (C) before the beginning of the poly(A) tract, was
hybridized to each of all three oligonucleotides separately and also
to an equimolar mix of the three. The signals from the two mis-
matching oligo(dT) primers were only 1.8% and 4.6% that of the
matching oligo(dT) primer. The signal from hybridization with a
mix of all three oligonucleotides was decreased in comparison to
the hybridization with only the oligo(dT)-deoxyguanine (dG)
primer, most likely due to the competition for hybridization sites.
water
mRNA Oligo Mix
10µl
7µl
3µl
Hybridize at 65°C
for 15 minutes.
Cool at room temperature
for 15 minutes.
Prepare reactionmaster mix.
Keep on ice.
Make 15µl aliquots.
Incubate at
37°C for 30 minutes.
Put on ice.
Add 15µl reactionmix to 100µl
L/L Reagent.
Read light
signal in
luminometer.
Add 5µl
hybridizationreaction.
Assay eachhybridization reactionin triplicate.
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Figure 2. Schematic of the Poly(A) mRNA Detection
System protocol.
3
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The use of anchored oligo(dT) primers permits the Poly(A) mRNA
Detection System to be dependent on the number of poly(A) tails
in the sample and less dependent on the mass of poly(A) orpoly(A) tail length. Table 2 shows the results when different
mRNAs are assayed. Thus light signal is dependent upon the num-
ber of mRNA molecules and not the mass of mRNA present.
LINEARITY AND SENSITIVITY
Because the system is dependent on the number of mRNA mole-
cules present, comparison of the number of light units produced by
known amounts of a standard mRNA can be used to estimate the
number of mRNA molecules present in samples. A purified 1.2kb
synthetic Kanamycin transcript is supplied with the system to be
used as the mRNA standard. Figure 3 shows the linearity and sen-
sitivity when this 1.2kb Kanamycin mRNA(c) is assayed using the
Poly(A) mRNA Detection System. The assay is linear for samples
containing up to approximately 2–4ng/ µl of Kanamycin mRNA
(or about 5 to 10fmol/ µl). At concentrations higher than this, the
signal begins to level off (Panel B). The sensitivity of the assay
was determined by further diluting the Kanamycin mRNA. The
limit of detection is <40pg/ µl of Kanamycin mRNA (or about
0.1fmol/ µl; Panel C; Table 3). We recommend using the system in
the linear range with sample concentrations of 40pg/ µl to 2ng/ µl.
Poly(A) mRNA Detection…continued
Table 1. Signal from Hybridization of Kanamycin mRNA withIndividual Oligonucleotides.
% of SignalOligo Light Units with Oligo(dT)-dG
Oligo(dT)-dC 3.605 1.8
Oligo(dT)-dA 9.220 4.6
Oligo(dT)-dG 199.0 100All Oligo(dT)s 130.0 65.3
Table 2. Signals from Equivalent Moles of Various mRNAs.1
LightUnits2 % Kanamycin
mRNA (LU) Signal LU/amol3 LU/pg
Kanamycin 130.0 100 0.13 0.33
Luciferase 110.5 85 0.11 0.19
Globin 180.7 139 0.18 0.88
All mRNAs4 122.2 94 0.12 –
1One femtomole of each mRNA was used per pyro/transphosphorylation reac-tion. The concentration in mass was determined by A260 values using a con-version factor of 40µg/ml = 1 O.D. unit. Known or estimated message lengthswere used to calculate grams/mole using the formula: #nucleotides × 330.One fmol of RNA corresponds to 396pg, synthetic Kanamycin mRNA (1.2kb,Cat.# C1381); 580pg, synthetic Luciferase mRNA (1.75kb, Cat.# L4561); and206pg Rabbit α- and β-Globin mRNA (estimated average of 625 bases,GibcoBRL.
2Light Unit values are the averages of three pyro/transphosphorylation reactions.3One attomole = 10–18mol.4Kanamycin, Luciferase and Globin mRNA, 1:1:1 molar ratio.
Figure 3. Linearity of the Poly(A) mRNA Detection
System. Kanamycin, 1.2kb, (0.5µg/µl) was diluted into water to
various concentrations within the linear range of the assay. The
concentration of the mRNA in the dilution is indicated on the X
axis. Ten microliters of each dilution were hybridized with the
Oligo Mix in a final volume of 20µl. After hybridization, 5µl
aliquots were added to triplicate pyro/transphosphorylation reac-
tions. The net average light units for the triplicate reactions are
plotted on the Y axis. Error bars represent ±1 standard devia-
tion. The best-fit line was generated by linear regression analy-sis. The absolute value for light units can vary dramatically with
the luminometer and reading conditions. Throughout this report,
light unit measurements were made in 8mm x 50mm luminome-
ter tubes using a Turner TD-20/20 Luminometer set at 52% sen-
sitivity. The signals were read for 15 seconds following a
three-second preread delay. The data for Panel C can also be
found in Table 3.
0 20 40 60 80 1000
20
40
60
80
100
N e t L i g h t U n i t s
Kanamycin mRNA (pg/ µl)
B.
A.
C.
0 1 2 3 4 5 60
500
1,000
1,500
2,000
2,500
3,000
3,500
4,000
N e t L i g h t U n i t s
Kanamycin mRNA (ng/ µl)
0 500 1,000 1,500 2,000
Kanamycin mRNA (pg/ µl)
0
200
400
600
800
1,000
1,200
1,400
1,600
1,800
N e t L i g h t U n i t s
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The comparison of a signal from a sample of unknown mRNA
content to the signal from the 1.2kb Kanamycin mRNA standard
gives an estimation of the number of poly(A) tails and polyadeny-
lated mRNA molecules in the sample. Alternatively, the concentra-
tion of polyadenylated mRNA in terms of mass can be estimated
by assuming an average mRNA length equal to the 1.2kb
Kanamycin mRNA or another length.
SPECIFICITY
Using the Poly(A) mRNA Detection System it is possible to dis-
criminate between RNA species that are not distinguishable by
spectrophotometry. In the following experiment, mRNA, total
RNA and tRNA were diluted to 4ng/ µl, the lowest concentration
that can be reliably measured using spectrophotometry. A 1:1 mix
of mRNA and total RNA was also prepared. As expected, all four
samples showed similar absorbances at 260nm (Figure 4). Results
of the Poly(A) mRNA Detection System were quite different. The
signal from the tRNA was only 0.7% that of the equivalent mass
of mRNA. The total RNA had a signal 4.6% that of the mRNA,
most likely indicating the percent of total RNA that is mRNA.
Also, the 1:1 mix had approximately 50% the mRNA signal.
This demonstrates the usefulness of this technology to measuremRNA levels in mRNA-enriched samples that may still have
rRNA contamination.
Table 3. Sensitivity of Poly(A) mRNA Detection System.
mRNASample Avg. NetConc. Light Light(pg/ µl) Units1 S.D. %C.V.2 Units3
0 16.77 0.43 2.6 0.0
20 33.20 0.50 1.5 16.25
40 51.31 0.78 1.5 34.54
60 62.75 0.53 0.8 45.98
80 75.56 5.16 6.8 58.79
100 98.38 1.58 1.6 81.61
1Light Unit values are the averages of three pyro/transphosphorylation reac-
tions. Light Unit values can vary with luminometer and reading conditions.2%C.V. = (100 × standard deviation)/average
3Net Light Units = Average Light Units – Light Units from the no mRNAsample control.
Figure 4. Discrimination of RNA species. Total RNA was
purified from mouse liver using SV Total RNA Isolation System
(Cat.#Z3100). mRNA was prepared from a portion of this total
RNA using the PolyATtract® mRNA Isolation System III (Cat.#
Z5300). Transfer RNA was isolated from calf liver. The concentra-
tions of the RNA preparations were determined spectrophotomet-
rically. They were then diluted to 4ng/ml. Equal volumes of the
mRNA and total RNA were combined to form a 50%:50% mix-
ture. Panel A: The concentration of nucleic acid was confirmed
spectrophotometrically.Panel B: The concentration of mRNA
in the preparations was determined using the Poly(A) mRNA
Detection System. The percentages above each bar represents
the percent of signal using mouse liver mRNA.
0.02
0.04
0.06
0.08
0.10
0.12
mRNA Total
RNA
1:1 tRNA mRNA Total
RNA
1:1 tRNA
100% 101%93% 93%
0
1,000
2,000
3,000
4,000
5,000100%
4.6%
46.6%
0.7%
A. B.
A 2 6 0
N e t L i g h t U n i t s
3 1 1 7 M
A 1 0_ 0
A
Table 4. Signal from rRNA1.
Avg. Avg. Calc. Conc.Conc. Light of nRNA3 Estimated
RNA (ng/ µl) Units2 (mg/ µl) %mRNA4
Kanamycin 2 2741
Total RNA 200 6816 4.97 2.5
2 68.6 0.50 2.5
Supernatant 200 3643 2.66 0.53
rRNA 2 30.8 0.022 0.50
1Total mouse liver RNA was purified using RNAgents® Total RNA IsolationSystem (Cat.# Z5110). This material was then passed through thePolyATtract® mRNA Isolation System (Cat.# Z5300) twice and the resulting
supernatant was assayed. An agarose gel confirmed the presence of tworRNA bands and the high quality of the preparations (data not shown). Theconcentration was determined spectrophotometrically and then the RNA wasdiluted to the indicated concentrations.
2Each sample was assayed in triplicate using the Poly(A) mRNA Detection System.
3Signals were compared to that from Kanamycin mRNA. The Kanamycin gener-ated a signal of 1370.5 light units/ng/ml and this was the conversion factorused to estimate the concentration of mRNA in the total RNA and supernatant.
4The percent of mRNA present in the starting material (e.g., 100 × 4.97/200)
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6 Promega Notes 77
To further investigate the signal generated by rRNA, a preparation
enriched for rRNA and relatively depleted of mRNA was prepared
by passing total RNA through the PolyATtract® mRNA Isolation
System twice. The resulting supernatant was assayed in the
Poly(A) mRNA Detection System. The signal from the super-
natant was reduced in comparison to signal from total RNA, sug-
gesting that signal from total RNA was generated by mRNA
molecules. The signal generated from the supernatant was only
0.5% that of an equivalent mass of mRNA.
DOSE RESPONSE CURVES WITH MRNA AND TOTALRNA Dose response curves using total RNA and Kanamycin mRNA
samples were generated for Figure 5. The signals from the mRNA
sample and the Kanamycin mRNA are consistent (Panel A). This
illustrates the usefulness of a 1.2kb mRNA as the standard when
measuring a population of heterogeneous mRNAs. A preparation
of total RNA was also tested (Panel B). Though the majority of
RNA (80–85%) in a total RNA preparation is rRNA (1,2), the sys-
tem does not produce significant signals with rRNA and tRNA and
thus allows for the measurement of mRNA even in the presence of
these species. The signal is produced by the 1–3% of RNA
expected to be mRNA.
REPRODUCIBILITY OF MRNA ESTIMATION
To determine the reproducibility of mRNA estimation, mouse liver
mRNA was prepared. The preparation was diluted to 400 and
40pg/ µl, and aliquots of each dilution were made and stored at
–20°C. Multiple experiments were performed. An aliquot of each
dilution was used per experiment and the concentration determined
using the Poly(A) mRNA Detection System with a Kanamycin
mRNA standard curve (Table 5).
SUMMARY
The Poly(A) mRNA Detection System is a new system for the
detection of picogram to nanogram mRNA. The technology relies
on a series of enzymatic reactions to generate ATP, which is thenmeasured using the light-emitting luciferase reaction. This system
is more sensitive and selective than other methods commonly used
to detect mRNA.
Poly(A) mRNA Detection…continued
Figure 5. Dose response curves with mRNA and total RNA.
Panel A: Total RNA was purified from mouse kidney using
RNAgents® Total RNA Isolation System (Cat.# Z5110). mRNA
was then purified from the total RNA using PolyATtract® mRNA
Isolation System III (Cat.# Z5300). The concentration of the
mRNA preparation was determined to be 121ng/µl by absorbance
at 260nm and a conversion factor of 40µg/ml per absorbance
unit. The mRNA was diluted to 5ng/µl and then to the concentra-
tions indicated above on the X axis. The hybridization and tripli-
cate reactions were carried out as described in the protocol.
Identical reactions were carried out using the 1.2kb Kanamycin
mRNA for comparison, and this data is also plotted. Panel B:
Total RNA was purified from mouse liver using SV Total RNA
Isolation System (Cat.# Z3100). The RNA, at 1.86mg/ml, was
diluted in water to the concentrations indicated on the X axis.
Note the scale difference between the two panels.
0 100 200 300 4000
2,000
4,000
6,000
8,000
10,000
N e t L i g h t U n i t s
Total RNA (ng/ µl)
0 1 2 3 4
0
1,000
2,000
3,000
4,000
5,000
6,000
7,000
N e
t L i g h t U n i t s
mRNA (ng/ µl)
Kanamycin
mRNA pool
A. B.
Table 5. Reproducibility of Poly(A) mRNA Detection System.1
Conc. of mRNA Avg. Conc.by O.D. (pg/ µl) (pg/ µl)2 S.D. %C.V.
400 377.6 92.6 24.5
40 35.5 6.4 17.9
1Total RNA was purified from mouse liver using SV Total RNA Isolation System(Cat.# Z3100). The preparation was then enriched for mRNA using the PolyATtract®
mRNA Isolation System III (Cat.# Z5300). No rRNA was visible by gel elec-trophoresis (data not shown). The concentration of the mRNA preparation was19ng/ µl by A260 using a conversion factor of 40µg/ml. An average length of
1.2kb was assumed to convert the concentration from moles to mass units.2The average concentration from eight experiments performed on six days bytwo operators using the Poly(A) mRNA Detection System.
2 9 9 4 M A 0 7_
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7
A CKNOWLEDGMENT
The authors wish to thank Brian Andersen, Mindy Bennett,
Francoise Chauvin, Gary Kobs, Brian McNamara and Terri
Sundquist for their efforts in the development and production of
this product.
REFERENCES
1. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular
Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor
Laboratory Press.
2. Lewin, B. (1997) Genes VI , Oxford University Press.
3. Deuscher, M.P. and Kornberg, A. (1969) J. Biol. Chem. 244,
3019–3028.
4. Kinney, J. et al. (1999) Promega Notes 73, 3–7.
5. Parks, R.E. and Agarwal, R.P. (1973) The Enzymes: Nucleoside
Diphosphokinases, Vol. 8, 3rd ed.
6. Moyer, J.D. and Henderson, J.F. (1983) Anal. Biochem. 131,
187–189.
PROTOCOLS
Poly(A) mRNA Detection System Technical Bulletin #TB282,
Promega Corporation.
(www.promega.com/tbs/tb282/tb282.html ) PolyATtract ® mRNA Isolation System Technical Manual #TM021,
Promega Corporation.
(www.promega.com/tbs/tm021/tm021.html )
RNAgents® Total RNA Isolation System Technical Bulletin #TB087,
Promega Corporation.
(www.promega.com/tbs/tb087/tb087.html )
SV Total RNA Isolation System Technical Manual #TM048,
Promega Corporation.
(www.promega.com/tbs/tm048/tm048.html )
Ordering Information
Product Size Cat.#
Poly(A) mRNA DetectionSystem 100 reactions K4040
Related Products
Product Size Cat.#PolyATtract® mRNAIsolation System IIIwith Magnetic Stand 15 isolations Z5300
RNAgents® Total RNAIsolation System scalable blots* Z5110
SV Total RNA IsolationSystem 50 preps Z3100
1.2kb Kanamycin PositiveControl RNA(c) 5µg C1381
Luciferase Control RNA(c,d) 20µg L4561
DNA Quantitation System(a) 100 reactions K4000
*The RNAgents® Total RNA Isolation System can process up to 1 gram of start-ing tissue or 108 cultured cells. The RNAgents® System also allows RNA isola-tion from as little as 5mg of starting tissue. A modified protocol has beendeveloped that allows RNA to be isolated in 90 minutes for use in RT-PCRapplications.
PolyATtract and RNAgents are trademarks of Promega Corporation and are
registered with the U.S. Patent and Trademark Office.
(a)Patent Pending.
(b)The PCR process is covered by patents issued and applicable in certain countries. Promegadoes not encourage or support unauthorized or unlicensed use of the PCR process.
(c)U.S. Pat. Nos. 4,966,964, 5,019,556 and 5,266,687, which claim vectors encoding a portion ofhuman placental ribonuclease inhibitor, are exclusively licensed to Promega Corporation.
(d)The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos.5,583,024, 5,674,713 and 5,700,673.
DONNA LEIPPE KEN LEWIS JOHN SHANNONHOUSE
JOHN SHULTZ
PRODUCT BIBIOLGRAPHY Look to our web site at: www.promega.com/ena/ for
citations on this new product in the coming months.
608-274-43301-800-356-9526
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