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RT² Nano PreAMP technology utilizes multiplex tandem PCR to pre-amplify gene-specific cDNA with
minimal bias. This kit is intended for pre-amplification of first strand cDNA from limited amount of total RNA
samples for gene expression analysis with our RT² Profiler PCR Arrays.
You can prepare enough cDNA from each RNA sample for gene expression
analysis on as many as 4 different PCR Arrays. Two simple steps are involved
in this kit:
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First strand cDNA synthesis
This kit provides enough reagents for synthesizing first strand cDNA
from 12 different RNA samples. Our RT² First Strand cDNA synthesis system in
this kit comes with a built-in external RNA control template that would
be detected by the Reverse Transcription Control (RTC) tests in the RT²
Profiler PCR Arrays. This allows the detection of any presence of
inhibitors of reverse transcription, ensuring the efficiency of the
first strand cDNA synthesis reactions.
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Pre-Amplification of cDNA for pathway-specific genes
Each first strand cDNA synthesis reaction from 1ng to 100ng of total RNA
can be amplified using 4 different sets of PCR Array-specific primer mixes,
allowing gene expression analysis on as many as four different PCR
Arrays.
The included Side Reaction Reducer eliminates the residual primers from pre-amplification,
enabling accurate detection on PCR Arrays.
To complete the PCR Array procedure, mix the pre-amplified
templates with one of our instrument-specific and ready-to-use RT² SYBR
Green qPCR Master Mixes. For the rest of PCR Array protocol, please see
How PCR Array Works protocol.
How to order:
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Select your PCR Arrays and
RT² SYBR Green qPCR Master
Mix
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Order RT² Nano PreAMP cDNA Synthesis Kit
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Select the matching RT² Nano PreAMP cDNA Synthesis
Primer Mix for your PCR Array
Important Note: Each RT² Nano PreAMP cDNA Synthesis Primer
Mix is PCR Array-specific and can only be used for the specified RT²
Profiler PCR Arrays. The first strand synthesis components and the RT² Nano
PreAMP reagents in this kit have been optimized to maximize the sensitivity of our RT²
Profiler PCR Arrays.
Performance
Figure 1: Increased Positive Call Rate-- First
strand cDNA was synthesized from 40ng mouse total RNA with (red bars) or
without (blue bars) pre-amplification with RT² PreAMP Master Mix and RT²
Nano PreAMP cDNA Synthesis Primer Mix (Mouse Inflammatory Cytokines &
Receptors). The unamplified and PreAMP amplified samples were then analyzed
on the Mouse Inflammatory Cytokines & Receptors RT² Profiler PCR Array
which consists of 84 pathway-specific assays plus 5 housekeeping genes, and
the threshold cycle values (Ct) were obtained. Genes with Ct≥35 were
considered to be "Absent". In the unamplifed sample, 75% of the
genes analyzed on the PCR Array were present. The positive call rate was
increased to 100% in the PreAMP amplified sample. Shown here are 22 genes
that are called "Absent" in the unamplified sample while these
genes are shown to be detectable in the PreAMP sample.
Figure 2: Unbiased Amplification Process-- Highly
Comparable ΔCt values between Pre-amplified and Unamplified cDNA from Human
Liver Tumor. First strand cDNA was synthesized from 5ng of human liver
tumor RNA. One-quarter of the RT product was then used for preamplification
with RT² PreAMP PCR Master Mix plus Human Cancer PathwayFinder RT² Nano
PreAMP cDNA Synthesis primer mix. Unamplified cDNA synthesized from 500ng
of the same liver tumor RNA sample was used as the control. PreAMP
amplified and unamplified cDNA samples were then analyzed on the Human
Cancer PathwayFinder RT² Profiler PCR Array which consists of 84
pathway-specific and 5 housekeeping gene assays and the threshold cycle
values (Ct) were obtained. ΔCt value for each gene was calculated by
subtracting the average Ct value of the five reference genes (B2M, HPRT1,
RPL13A, GAPDH and ACTB) on the PCR Array from the Ct value of the gene of
interest. The concordance of Ct values between pre-amplified and
unamplified samples was evaluated by regression analysis. Data points with
Ct≥35 were considered to be absent genes and were excluded from the
analysis. The dashed line on each graph represents the ideal slope of 1.0.
The solid lines show a linear regression fit with the R2 and slope
indicated in the graph. The high correlation between pre-amplified and
unamplified cDNA was also obtained from Human Universal RNA Sample (data
not shown).
Figure 3: Faithfully Amplified Biology- Highly
Comparable Gene Expression Fold Change Results between Pre-amplified and
Unamplified Samples. First strand cDNA was synthesized from 1ng human liver
tumor RNA or universal RNA. One-quarter of each RT product was then used
for preamplification with RT² PreAMP PCR Master Mix plus Human Cancer
PathwayFinder RT² Nano PreAMP cDNA Synthesis primer mix. Unamplified cDNA
synthesized from 500ng of each corresponding RNA sample was used as the
control. PreAMP and unamplified cDNA samples were then analyzed on the
Human Cancer PathwayFinder RT² Profiler PCR Array which consists of 84
pathway-specific and 5 housekeeping gene assays, and the fold change in
gene expression between liver tumor and universal RNA for each gene was
obtained using the ΔΔCt method. The dashed line represents the ideal slope of
1.0. The solid lines show a linear regression fit with the R2 and slope
indicated in the graph. The fold change results obtained using preamplifed
cDNA generated from 5ng or 50ng of liver tumor RNA or universal RNA showed
similar or better correlation when compared to the unamplified samples,
with R2=0.95 and R2=0.97, respectively (graphs not shown).
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