> Protocol for PolyA mRNA Detection
for PolyA mRNA Detection
concentration and quality of mRNA is an important
parameter to consider prior to generating cDNA probes
for microarray analysis. Conventional spectrophotometric
methods cannot be used to distinguish contaminating
rRNA and tRNA species in a total RNA preparation.
A sensitive and specific Poly(A) mRNA Detection System
has been developed, based on the pyrophosphorylation
activity of DNA Polymerase I (Klenow; Exonuclease
minus) that acts only on DNA strands perfectly matched
at their 3-termini. Anchored oligo(dT) primers hybridize
to poly(A) mRNA, followed by a coupled pyrophosphorylation
and transphosphorylation reaction, resulting in the
production of ATP in a quantity proportional to the
amount of poly(A) tails present. ATP concentration
is then measured in the highly sensitive TD-20/20
Luminometer using recombinant luciferase. A standard
curve is generated using a purified 1.2kb synthetic
Kanamycin transcript to estimate the number of mRNA
molecules present in unknown samples.
Luminometer with 12 mm test tube holder and 12x50
mm test tubes (P/N 2020-001)
10-100 µL volume pipetter and RNAse-free tips
1-10 µL volume pipetter and RNAse-free tips
mL and 1.5 mL Microcentrifuge tubes
or latex gloves
C and 65° C water bath or heating block
sterile, deionized water
mRNA Detection System (Promega Cat. # K4040) which
1 mL DNA Polymerase 10X Buffer
50 µL Sodium Pyrophosphate (40 mM)
150 µL ADP (20 µM)
15 µL Nucleoside 5´ Diphosphate Kinase (NDPK)
1000 units DNA Polymerase I, Klenow Fragment Exonuclease
300 µL mRNA Detection Oligo Mix (1 µg/µL)
20 µL 1.2kb Kanamycin mRNA (0.5 µg/µL)
1 vial Luciferase/Luciferin Reagent (luciferase Reagent)
1 vial ENLITEN ® Reconstitution Buffer (12 mL)
Gloves should be worn during the reconstitution of
luciferase reagent and while performing the assay
to avoid contamination with ATP and RNAses. Luciferase
enzyme activity will decline with multiple freeze-thaw
on instrument and allow to warm up for at least
period: 3 seconds
period: 15 seconds
of replicates: 1
Luciferase Reagent Preparation
the vial of Luciferase reagent with 12 mL of the
ENLITEN Reconstitution Buffer.
invert the vial several times to dissolve the contents,
and allow the reconstituted reagent to equilibrate
to room temperature for at least 60 minutes.
luciferase reaction is temperature-sensitive; therefore,
ensure that the temperature of the reagent does
not fluctuate during a set of readings.
the reconstitution step is completed, the prepared
reagent may be kept at room temperature (up to 8
hours). Unused reconstituted reagent should be dispensed,
as single-use aliquots, into sterile microfuge tubes
and frozen at 20°C.
Sample Preparation: Hybridization
· 10 µL of sample to be tested
· 3 µL of Oligo Mix
· 7 µL of RNase-free, sterile, deionized
a negative control also prepare hybridization reactions
· 3 mL of Oligo Mix
· 17 mL of RNase-free, sterile, deionized
samples and standards at 65°C for 15 minutes.
the tubes to cool by placing them at room temperature
for 15 minutes.
at 12,000 x g for 10 seconds to collect condensation.
Reaction Mix Preparation
µL Sterile, deionized water
µL 10X DNA polymerase buffer
µL sodium pyrophosphate
µL NDPK enzyme solution
µL Klenow exonuclease minus for a total
volume of 15.0 µL
E. Pyrophosphorylation/ Transphosphorylation Reaction
15 µL of the above Reaction mix into labeled
5 µL of each hybridization reaction to each
of the tubes. Vortex gently. Assay each reaction
at 37°C (in a heating block or water bath) for 30
minutes. All samples must be incubated for the same
period of time.
100 µL of the reconstituted luciferase reagent
into luminometer tubes, preparing one tube per reaction.
the 30-minute incubation, place reactions on ice
and analyze as soon as possible (within 2 hours).
15 µL from one reaction mix and place into
100 µL of reconstituted luciferase reagent.
read the sample once it has been added to the
luciferase reagent, as the enzyme rapidly begins
to utilize the ATP.
steps 6 & 7 above until all samples are read.
Calculation of mRNA Concentration
triplicate sample values.
the averaged value for the blank reaction, i.e.,
no RNA, from the values obtained from samples. This
yields net light units.
the standard curve by plotting mRNA concentration
versus net light units and drawing a best-fit line.
the concentration of the unknown samples by comparing
the net light units (LU) seen for these samples
against those seen for the standard curve.
M.P. and Kornberg, A. (1969) Enzymatic synthesis of
deoxyribonucleic acid. The pyrophosphate exchange
and pyrophosphorolysis reactions of deoxyri-bonucleic
acid polymerase. J. Biol. Chem.244, 3019.
J.D. and Henderson, J.F. (1983) Nucleoside triphosphate
specificity of firefly luciferase. Anal. Biochem.131,
J., Fritsch, E.F. and Maniatis, T. (1989) Molecular
Manual, 2nd ed., Cold Spring Harbor Laboratory Press.
B. (1997) Genes VI, Oxford University Press.
Poly(A) mRNA method was adapted from a Note (#77)
written by Promega Corporation scientists. Data
obtained from this Note indicates that the assay
is linear over the 20pg/ mL to 2ng/ mL concentration
range. Further details about the system can be found
on their website.
2800 Woods Hollow Road
Madison, WI 53711