Download RNA Amplification and cDNA Synthesis for qRT

MessageBOOSTER™ Kit
RNA Amplification and cDNA Synthesis for qRT-PCR Directly
from a Single Cell without RNA Purification
Judith E. Meis, Anupama Khanna, and Jim Pease, EPICENTRE Biotechnologies
Introduction
Quantitative reverse-transcription PCR (qRT-PCR) gene expression studies from very small number of cells (e.g.,
1-1,000 cells) can be challenging for a variety of reasons:
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Difficulty in purifying extremely small amounts of total RNA.
A limited number of reactions can be performed, and very few transcripts analyzed, per sample.
Lack of sensitivity, especially when detecting low- and medium-abundance messages.
The need to collect samples often.
The inability to archive samples for future use.
The new MessageBOOSTER™ cDNA Synthesis from Cell Lysates Kit eliminates these difficulties by enabling RNA
amplification and cDNA synthesis directly from cell lysates, without the need to purify total RNA. Here we demonstrate
that a MessageBOOSTER Kit reaction produces enough cDNA directly from the lysate of a single cell for thousands of
qPCRs, and that the cDNA produced enables sensitive detection of even low-abundance transcripts.
Methods
An overview of the process for the MessageBOOSTER
cDNA Synthesis from Cell Lysates Kit is illustrated in
Fig. 1. Briefly, cells are lysed and the poly(A) RNA (mRNA)
in the crude, whole-cell lysate is amplified by in vitro
transcription. The amplified RNA is converted to cDNA,
and the cDNA diluted up to 1,000-fold for qPCR. The
single-day reaction will efficiently produce cDNA directly
from lysates of 1 to 1,000 cells.
Cell collection and lysis
Cultured normal rat kidney (NRK) or HeLa cells were
collected, counted, and placed into sterile 0.5-ml
microcentrifuge tubes on ice. The cells were pelleted
at 4°C at 500-1,200 x g for 5 minutes, and the culture
medium was carefully removed. The cells were resuspended
in 3 µl of QuickExtract™ RNA Extraction Solution
(provided in the kit; see also p. 14) and lysed either by:
i) freezing in liquid nitrogen and then thawing for 2
minutes at room temperature; or ii) vigorous vortex
resuspension for 1 minute. The cell lysates were placed on ice.
RNA amplification and cDNA synthesis
The MessageBOOSTER Kit reaction was performed
directly in the cell lysates. As shown in Fig. 1, an oligo(dT)
primer containing a T7 promoter and MMLV Reverse
Transcriptase were used to synthesize first-strand
cDNA from poly(A) RNA. After second-strand cDNA
synthesis, a high-yield in vitro transcription reaction
was used to amplify the poly(A) RNA (mRNA). The
in vitro transcription reaction generates thousands of
RNA molecules for every molecule of double-strand (ds)
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cDNA template, and thus serves as a powerful engine
for amplifying the poly(A) RNA. Following in vitro
transcription, the samples were treated with DNase I to
remove the ds cDNA template, as well as a large portion
of genomic DNA. The amplified RNA was purified
using an RNA Clean & Concentrator™-5 column (Zymo
Research) and then reverse-transcribed, using random
primers, into cDNA in a final volume of 12.5 µl. cDNA
produced by the MessageBOOSTER Kit reaction was
designated as “MessageBOOSTER cDNA.”
Control reactions
cDNA was synthesized directly from cell lysates without
an RNA amplification step. This cDNA synthesis process,
designated as the “cDNA-from-cells” procedure, was
performed by reverse transcription using an oligo(dT)
primer in a 20-µl reaction. In some cases, a commercial
cDNA-from-cells–type kit was used.
A MessageBOOSTER Kit reaction was performed without
the initial reverse transcriptase. This control, designated
as the “No-RT” control, was performed in order to
evaluate the level of residual genomic DNA in the
samples after the MessageBOOSTER Kit reaction.
Real-time PCR
The cDNA produced by the MessageBOOSTER Kit
reactions was diluted 1:10 and 1:1,000 in water for
subsequent qPCR. A 1-µl aliquot of the diluted cDNA
was used for each 25-µl qPCR. qPCR was performed
using the TAQurate™ GREEN Real-Time PCR System
(EPICENTRE). Volume 16-2 • June 2009
MessageBOOSTER™ Kit
Results
Benefit of the MessageBOOSTER RNA amplification step
for small samples
The MessageBOOSTER Kit uses a linear RNA amplification
step that generates >1,000 RNA molecules from each
molecule of poly(A) RNA in the cell lysate. In addition, the
RNA amplification includes a DNase treatment step that
significantly reduces the genomic DNA in the lysate.
cDNA was produced from a single HeLa cell using the
MessageBOOSTER kit and the cDNA-from-cells procedure
(which does not include an RNA amplification step). qPCR
was performed using 1 µl of undiluted cDNA produced by
each reaction and a primer pair for the LDHA transcript.
The No-RT control reaction was performed in order to
assess the amount of residual genomic DNA present after
a MessageBOOSTER kit reaction. As shown in Fig. 2A, the
LDHA transcript was readily detected using cDNA produced
Cultured Cells
Cell Lysis
Cell Lysis Solution
Cell Lysate
No RT
1 cell
No RNA
amplification
T7-Oligo (dT) Primer
TTTT
T7
Cycle
AAAA
TTTT
T7
AAAA
TTTT
T7
T7
Round 1
Second-Strand
cDNA Synthesis
B
In Vitro
Transcription
UUUU
Round 2
First-Strand
cDNA Synthesis
5 cells
–d(RFU)/dT
Round 1
First-Strand
cDNA Synthesis
Relative Fluorescence Units
A
aRNA (cRNA)
Random Hexamers
T,°C
UUUU
AAAA
RNase H
AAAA
cDNA
PCR
(provided by user)
Figure 2. A MessageBOOSTER™ Kit reaction enables sensitive and specific
detection of low-abundance transcripts. A, qPCR detection of the LDHA
transcript in lysates from five HeLa cells (purple) and a single HeLa cell using
cDNA produced by a MessageBOOSTER Kit reaction (blue), by a cDNA-fromcells procedure, without benefit of an RNA amplification step (green), and
from a No-RT control reaction (black). B, Melt-curve analysis of the qPCR
products revealed that the MessageBOOSTER cDNA produced an authentic
LDHA amplicon, while the cDNA-from-cells procedure and No-RT control
reaction generated nonspecific amplification products; these results were
confirmed by gel electrophoresis (not shown).
Figure 1. An overview of the procedure for the MessageBOOSTER™
cDNA Synthesis from Cell Lysates Kit. A kit reaction amplifies the poly(A)
RNA (mRNA) directly from a crude cell lysate without the need for RNA
purification. The amplified RNA is then reverse-transcribed to cDNA that can
be diluted up to 1,000-fold for qPCR.
Volume 16-2 • June 2009 www.EpiBio.com
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Relative Fluorescence Units
MessageBOOSTER™ Kit
1:100
1:1,000
1:10
undiluted
NRK-cell lysate. Thus, a MessageBOOSTER Kit reaction can
produce sufficient cDNA for >1,000 qPCRs from a single-cell
lysate. The number of qPCRs that can be performed using
cDNA produced by the MessageBOOSTER Kit is thus
dependent on the number of cells and the abundance of the
transcript(s) of interest.
Conclusions
Cycle
Figure 3. A MessageBOOSTER™ Kit reaction produces sufficient cDNA from
a single-cell lysate for thousands of sensitive qPCRs. qPCR was performed
using undiluted (red), 1:10 diluted (green), 1:100 diluted (blue), and 1:1,000
diluted (purple) cDNA from a lysate of a single NRK cell. The low-abundance
PBGD transcript was readily detected.
by a MessageBOOSTER Kit reaction from five cells (CT = 19)
and from a single cell (CT = 22). The cDNA-from-cells and
the No-RT control produced only nonspecific signals, as
determined by the melting curve analysis in Fig. 2B. The
melting temperature of the nonspecific products was significantly
different from that of the LDHA-specific amplicon. Thus, the
RNA amplification step, included in the MessageBOOSTER
Kit reaction, enables sensitive and specific detection of
transcripts from a single-cell lysate and reduces the amount of
genomic DNA to undetectable levels. In contrast, the cDNAfrom-cells procedure yielded cDNA that did not detect the
desired transcript in qPCR.
Sensitive detection of low-abundance transcripts
We further investigated the detection limit of a low-abundance
transcript using cDNA produced by a MessageBOOSTER Kit
reaction from a single-cell lysate. MessageBOOSTER cDNA,
produced from a single NRK-cell lysate was diluted 1:10, 1:100,
and 1:1,000, prior to qPCR. As shown in Fig. 3, cDNA diluted
1:1,000 enabled detection (CT = 29) of the low-abundance
PBGD transcript. Melting curve analysis (data not shown)
confirmed that the correct amplicon was produced. Thus, a
MessageBOOSTER Kit reaction enables sensitive detection of a
low-abundance transcript using up to a 1,000-fold dilution of
the cDNA produced.
The data presented demonstrate that cDNA produced by a
MessageBOOSTER cDNA Synthesis from Cell Lysates Kit
reaction eliminates the difficulties frequently encountered
when performing qPCR gene expression studies from very
small numbers of cells. We have shown that:
• The kit enables the user to amplify the mRNA and
convert it to cDNA directly from a cell lysate without the
need to purify total RNA. RNA amplification and cDNA
synthesis directly from cell lysates eliminates the difficulty
and irreproducibility of purifying RNA from very small
numbers of cells. Because the RNA amplification and
cDNA synthesis reactions are performed directly in the cell
lysates, there is no loss of RNA as can occur when purifying
RNA from very small samples.
• A MessageBOOSTER Kit reaction, which includes a
linear RNA amplification step, significantly improves the
sensitivity of detecting even low-abundance transcripts
from as little as one cell.
• A single MessageBOOSTER Kit reaction produces enough
cDNA for thousands of qPCRs. More qPCRs per sample
means that samples are collected less frequently and that
there is plenty of cDNA produced to enable the user to
archive the sample for future use.
Cat. #
Quantity MessageBOOSTER™ cDNA Synthesis from Cell Lysates Kit
MBCL90310
10 Reactions
Are you performing qRT-PCR studies using purified total RNA?
Try the original MessageBOOSTER™ cDNA Synthesis Kit for qPCR
(Catalog Numbers MB060110, MB060124) which enables hundreds of
sensitive qRT-PCRs from as little as 10 pg of purified total RNA. Visit
www.EpiBio.com for details.
Yield of amplified cDNA/number of qPCRs obtained
The final step of a MessageBOOSTER Kit reaction converts
amplified RNA to cDNA by reverse transcription in a
reaction volume of 12-15 µl. As shown in Fig. 3, 1:10, 1:100,
and 1:1,000 serial dilutions of the cDNA enabled sensitive
detection of the low-abundance PGBD transcript from a single
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Volume 16-2 • June 2009