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Technical Report #1
Ovation™ RNA Amplification System
A Comparison of Gene
Expression Levels in
Non-Amplified and
Amplified RNA
BACKGROUND
Real-time quantitative PCR (QPCR)
to examine the accuracy, reproducibility
from Integrated DNA Technologies (IDT)
and sensitivity of the Ribo-SPIA
according to Primer Express® 2.0
amplification process used in the
(Applied Biosystems) rules or purchased
™
Ovation RNA Amplification System.
from Assays-on-Demand™ (Applied
Using real-time PCR, we examined
Biosystems). Contact NuGEN Technical
these parameters by measuring
Services ([email protected]) for
expression levels of a panel of genes
information regarding primer and probe
in both amplified and non-amplified
sequences and experimental design.
RNA preparations.
technology measures the relative
expression levels of specific genes
RESULTS AND CONCLUSIONS
MATERIALS AND METHODS
across many samples. Recommendations
Using 20 ng starting total RNA, the
for each QPCR reaction include a
Total RNA samples used in this experi-
Ovation™ RNA Amplification System
minimum of 10,000 template copies or
ment were Universal Human Reference
yielded an average of 2.0 µg of cDNA
1-100 ng of total RNA (Applied Biosystems
RNA (UHR, Stratagene cat. #74000), a
product with a standard deviation of 0.1
Protocol). Thus, to study a series of
RNA mixture from 10 cancer cell lines,
µg. This yield was within the expected
gene transcripts in replicates, microgram
and human skeletal muscle (SKMU,
range of 1.5 to 2.0 µg of cDNA from 5
quantities of total RNA may be required.
BD Clontech cat. #636534).
to 100 ng of starting total RNA.
limited and this restricts the number of
Triplicate reactions of each sample type
An assessment was made of the fidelity
gene transcripts that can be studied.
were used in both amplified and non-
of amplification of differentially expressed
Compounding the matter, analyses of
amplified reactions. Twenty ng of starting
genes. QPCR Ct values were measured
low abundance transcripts require large
total RNA were amplified with the
for 39 gene transcripts in both amplified
amounts of total RNA input for each
Ovation™ RNA Amplification System
and non-amplified UHR and SKMU RNA.
QPCR measurement.
(NuGEN, cat. #2200-48). For the non-
The Ct value reflected the expression of
Often the size of the starting sample is
amplified cDNA product, 4 µg of total
a specific transcript in a given RNA
One approach to enhancing the sensitivity
RNA was reverse transcribed using
preparation. The difference in expression
of QPCR analyses is pre-amplification of
RETROscript™ (Ambion, cat. #1710).
of a gene between RNA preparations
total RNA; enriching the amount of mRNA
Both cDNA products subsequently were
(UHR vs. SKMU) was determined by
species while successfully maintaining
diluted 1:10 in 1 x TE prior to QPCR.
subtracting the Ct value in UHR RNA
relative gene expression levels compared
Eight ng of amplified cDNA and 20 ng
from the Ct value in SKMU RNA. This
to the starting material. Additionally,
of RNA equivalent (non-amplified) was
difference in Ct values (∆Ct) reflects the
pre-amplifying total RNA addresses
added to each QPCR reaction. Real-time
differential expression of a gene between
sample limitations, creating opportunities
PCR was performed on a panel of 39
the two RNA preparations. Delta Ct values
for researchers to answer questions
genes. Genes were chosen for varying
(and hence differential expression) were
previously limited by sample size.
levels of gene expression and primer
calculated for all 39 gene transcripts in
positions at varying distance from the
amplified and non-amplified RNA. A
Up to 10,000 fold amplification of mRNA
poly A tail. Data was analyzed according
strong correlation of resultant ∆Ct values
species in a total RNA preparation has been
to Applied Biosystems User Bulletin #2.
(which are a measurement of differential
described for RiboSPIA™ amplification
gene expression) was obtained with
QPCR primers and probes (Dual
amplified and non-amplified RNA
results from a series of studies designed
Labeled DNA probes) were synthesized
(Figure 1), with a linear regression
DISCOVERY
technology. This report describes the
imagine more from less
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Technical Report #1
Ovation™ RNA Amplification System
correlation coefficient (R2) of 0.97.
and two QPCR assays per sample. The
that the QPCR primer/ probe sets
These results demonstrate that differen-
Ct results for the panel of 39 genes are
(amplicons) were from the 3’, poly A tail.
tial expression of genes (spanning low
shown in Figure 3. The mean Cts were
Similar Ct values were obtained between
to high abundance levels in both UHR
plotted with corresponding standard
amplified and non-amplified RNA,
and SKMU RNA, as measured by real-
deviations (Figure 3). (Note that the
regardless of amplicon location. These
time PCR,) was maintained after
standard deviations were determined
data demonstrate that with amplified
amplification with the Ovation RNA
and plotted for all gene transcripts;
cDNA product, QPCR primer and probe
Amplification System
however, due to the high level of repro-
sets can be chosen up to at least
ducibility, many of the standard deviation
1.5 kb from the 3’ poly A tail.
A comparison of expression levels in
determinations were very small and
amplified and non-amplified human
subsequently difficult to visualize in
Maintenance of relative gene abundance
skeletal RNA was made. Individual Ct
Figure 3). The data shows that high,
levels across a broad spectrum of genes
values were determined for each of the
medium and low expressing transcripts
is essential in any gene expression
39 genes in amplified and non-amplified
can be confidently amplified with an
methodology requiring amplification of
RNA. The resulting Ct values were plot-
average, coefficient of variation (CV)
mRNA. The Ovation™ RNA Amplification
ted and demonstrated a high level of
of 1.9%, for the 39 genes.
System maintains relative gene
correlation with a R2 value of 0.90
expression assuring that expression data
(Figure 2). Regression analysis of Ct
QPCR amplicons can be designed at
following amplification is representative
values for amplified and non-amplified
various locations on a gene transcript.
of the gene expression in the starting
RNA demonstrated a linear amplification
In the current study, amplicon locations
material. Furthermore, pre-amplification
profile with a slope of 0.97. These results
for the 39 genes were chosen up to
enhances the abundance of all mRNA
demonstrate linear amplification over
1.5 kb from the 3’ poly A tail. Figure 4
species, which results in an increased
a broad dynamic range.
depicts the Ct values for a representative
sensitivity with subsequent QPCR
set of the gene transcripts in amplified
analyses. This increase in sensitivity
Reproducibility of amplification was
and non-amplified samples. The Ct values
reflects the enrichment of mRNA species
determined using triplicate RNA samples
are plotted as a function of the distance
in the sample after amplification.
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Figure 1. Linear correlation of
differential gene expression in
non-amplified and amplified RNA
Non-Amplified cDNA (∆Ct UHR - SKMU)
R2 = 0.97
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Differences in differential gene expression between
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UHR and human skeletal muscle RNA are presented
as ∆Cts. For non-amplified product, 4 µg of total
0
-25
-20
-15
-10
-5
RNA was reverse transcribed prior to QPCR.
0
-5
5
10
15
20
Approximately 20 ng of reverse transcribed RNA
equivalents was loaded into each PCR well. For
amplified product, 20 ng of total RNA was used to
-10
produce SPIA™ amplified cDNA. The product was
diluted 1:10 to 4 ng/µl of cDNA product and 2 µl
-15
(8 ng) was loaded into each QPCR reaction. The
∆Ct values for amplified RNA and non-amplified RNA
-20
Amplified cDNA (∆Ct UHR - SKMU)
were plotted on the x-and y-axis, respectively.
A high level of correlation (R2 = 0.97) was obtained.
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Technical Report #1
Ovation™ RNA Amplification System
50
Figure 2: Correlation of Ct values
between amplified and non-amplified
skeletal muscle RNA
2
R = 0.90
Non-Amplified cDNA Ct
40
Ct values were determined for a panel of genes in
Ribo-SPIA™ amplified and non-amplified skeletal
muscle RNA. Amplified versus non-amplified values
30
were compared. The location of the linear regression
line (black) with a slope of 0.97 indicates linear
amplification lowers Ct values for amplified
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compared to non-amplified material.
10
0
0
10
20
30
40
50
Amplified cDNA Ct
40
Figure 3: Reproducibility of amplification
in human skeletal muscle RNA
Threshold Cycle (Ct)
Mean Ct values and standard deviations of the
39 genes are shown. Each point represents an
average of 6 data points obtained from 3 separate
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amplification reactions, which were followed up
with duplicate QPCR reactions.
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10
0
10
20
30
40
Gene Number
Figure 4: Primer and probe sets can be
chosen up to 1.5 kb from poly A tail
Threshold Cycle (Ct)
40
Locations of QPCR amplicons for amplified and
30
non-amplified cDNA for 12 different gene transcripts
are depicted. The x-axis represents the number of
bases the amplicon is located from the poly A tail.
20
Ct values were determined for both amplified and
non-amplified cDNA.
10
0
0
250
500
750
1000
1250
1500
Number of Bases from the Poly A Tail
Amplified
Non-Amplified
NuGEN Technologies, Inc. • 821 Industrial Road, Unit A • San Carlos CA 94070 USA • Toll-free 888.654.6544 • Tel 650.590.3600 • www.nugeninc.com
© Copyright 2004, NuGEN Technologies, Inc. This product and methods using this product are covered by pending patent applications including the following patent publications:
WO 02/072772: US2003/0017591 A1. NuGEN™, Ovation™, Ribo-SPIA™, and SPIA™ are trademarks or service marks of NuGEN™ Technologies, Inc.
All other marks appearing in these materials are marks of their respective owners.
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