EPICENTRE Forum 14-2

EPICENTRE® Biotechnologies Forum
A Simple Enzymatic Process for Generating mRNA-Enriched
Poly(A)-Tailed RNA from Total RNA Samples
Merriann Carey, EPICENTRE Biotechnologies
FIG 1. Overview of the mRNA-ONLY Prokaryotic
mRNA Isolation Kit with Poly(A)-Tailing.
Terminator Exonuclease digests the 16S and 23S
rRNAs in prokaryotic RNA to yield an enriched mRNA
preparation. The remaining RNA molecules are then
polyadenylated at the 3′-end using A-Plus Poly(A)
Polymerase in the presence of ATP.
Introduction
Metagenomics has spurred a growing
interest in using environmental mRNA or
transcriptomes to correlate genetic potential
with patterns of microbial activity. However,
progress has been hindered in part by the
difficulties of working with bacterial mRNA.
Bacterial mRNA generally lacks the poly(A)
tail, which makes the isolation and analyses
of eukaryotic messages fairly straightforward.
Moreover, bacterial rRNAs, like their
eukaryotic counterparts, can make up 80%
or more of total RNA, overwhelming samples
with background signals. EPICENTRE’s new
mRNA-ONLY Prokaryotic mRNA Isolation
Kit with Poly(A)-Tailing overcomes these
limitations with a simple two-step enzymatic
process that generates mRNA-enriched
poly(A)-tailed RNA from total cellular RNA
samples. The resulting polyadenylated mRNA
can then serve as a template for synthesizing
cDNA for quantitative RT-PCR, cDNA
library construction, and array analysis.
Production of mRNA-enriched
poly(A)-tailed RNA
The two-step enzymatic process provided
by the mRNA-ONLY Prokaryotic mRNA
Isolation Kit with Poly(A)-Tailing is shown
in FIG 1. EPICENTRE’s Terminator
5′-Phosphate-Dependent Exonuclease* is
used to digest ribosomal RNAs having a
5′-monophosphate. This processive 5′–3′
exonuclease does not digest RNA having a
5′-triphosphate, a 5′-cap structure (present on
most eukaryotic mRNAs), or a 5′-hydroxyl
group. Consequently, Terminator Exonuclease
can be used to isolate prokaryotic mRNA with
minimal carryover of 16S and 23S rRNA. 1
The remaining RNA molecules are then
polyadenylated at the 3′-end using A-Plus
Poly(A) Polymerase in the presence of ATP.
First-Strand cDNA Synthesis
As a simple test case, mRNA-enriched
poly(A)-tailed E. coli RNA, prepared using
the mRNA-ONLY Prokaryotic mRNA
Isolation Kit with Poly(A)-Tailing, was reverse
transcribed using an oligo(dT) primer and
MMLV Reverse Transcriptase (MMLV RT).
The resulting cDNA was used for real-time
PCR (qPCR) without additional treatment
or purification. qPCR was performed using
the TAQurate GREEN Real-Time PCR
MasterMix. a Control reactions were also
performed under the same conditions except
that MMLV RT was omitted.
FIG 2. Analysis of mRNA-enriched poly(A)-tailed
E. coli RNA by real-time RT-PCR. Each amplification
plot is color coded and the corresponding gene designations
are listed in Table 1.
Target ETCN C T
fliC 51 17.2
rplK 51 17.9
pal 18 18.1
cyoA 16 20.6
ilvE 4 22.0
basR 2 22.4
fecD 0.3 25.9
yafL 0.1 27.0
Table 1. Comparison of
Estimated Transcription
Copy Number (ETCN)
and Cycle Threshold (C T
)
Values.
As shown in FIG 2 and Table 1, the average
cycle threshold (C T
) value for each of the
eight targets tested correlates well with the
estimated transcription copy number (ETCN)
derived from E. coli microarray data. 2 Thus,
a highly expressed gene such as fliC with
an ETCN of 51 has a correspondingly high
real-time RT-PCR signal (low C T
value). Even
the 3-fold difference in ETCN levels for the
rare sequences fecD and yafL was represented
by reproducible differences in C T
values.
Preservation of relative mRNA
abundance levels
To evaluate the effect of Terminator
Exonuclease and A-Plus Poly(A) Polymerase
on the relative abundance of the eight mRNA
transcripts listed in Table 1, real-time RT-PCR
was performed before and after treatment with
these two enzymes. The “untreated” cDNA
was produced from total E. coli RNA using
MMLV RT and random nonamer primers.
The “treated” cDNA was produced as described
in the previous section. The results shown in
FIG 3 are expressed as the difference in C T
values (∆C T
) between the target message and
the normalizer (pal) for untreated and treated
samples. The high correlation coefficient
of R 2 =0.935 demonstrates that the cDNA
produced from the mRNA-enriched poly(A)-
tailed RNA conserved the relative abundance
of different RNA species.
Amplification of environmental mRNA
To validate the efficacy of Terminator
Exonuclease and A-Plus Poly(A) Polymerase
in the analysis of environmental RNA, a
fresh sample of dog feces, known to be rich
in bacteria, was used. Initially, DNA isolated
from dog feces (using the MasterPure DNA
Purification Kit) was screened by end-point
PCR with the same set of primers used to
amplify the E. coli genes listed in Table 1.
Four of the eight PCR reactions (cyoA, pal,
rplK, and yafL) yielded amplicons of the
expected size (data not shown).
Total RNA isolated from the same fecal
sample as the genomic DNA, was then treated
sequentially with Terminator Exonuclease and
A-Plus Poly(A) Polymerase (FIG 4). Since the
amount of total RNA from environmental
samples is likely to be small, the poly(A)-tailed
RNA produced by the Terminator Exonuclease
and A-Plus Poly(A) Polymerase treatment
was amplified using the MessageBOOSTER
cDNA Synthesis Kit for qPCR b before further
Relative Expression (∆C T )
10
8
6
4
2
0
-2
yafL fecD basR ilvE cyoA rplK fliC
RNA Target
Untreated
Terminator Exonuclease +
A-Plus Poly(A) Polymerase
FIG 3. Treatment of E. coli RNA with Terminator
Exonuclease and A-Plus Poly(A) Polymerase maintains
relative mRNA abundance levels as determined
by real-time RT-PCR.
c1930705
18
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Volume 14 • Number 2