mrna detection

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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

2 7 1 8 M B 0 6_

0 A



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.

2 9 8 2 M A 0 6_

0 A

Figure 2. Schematic of the Poly(A) mRNA Detection

System protocol.

3



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


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


2 9 8 0 M B 0 6_

0 A

4 Promega Notes 77



5

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


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)



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


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_

0 A



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,


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

RNAgents® Total RNA Isolation System Technical Bulletin #TB087,


(www.promega.com/tbs/tb087/tb087.html )

SV Total RNA Isolation System Technical Manual #TM048,


(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

[email protected]

Not pictured:

SCOTTWEATHERBEE

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