RT² FFPE RNA Extraction Kit
- Simplified RNA Isolation: No Xylene needed for the de-paraffinization
- Improved RNA Recovery: More effectively reverses formalin cross-links than
- Greater Real-Time PCR Sensitivity: Lowers real-time PCR threshold cycle
RT² First Strand cDNA Synthesis and
Pre-amplification of cDNA
The RT² 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 fragmented
total RNA of FFPE 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:
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.
Pre-Amplification of cDNA for pathway-specific genes
Each first strand cDNA synthesis reaction 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
the pre-amplification, reaction, preventing non-specific reactions, and 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:
Order RNeasy FFPE Kit and the RT² PreAMP cDNA Synthesis Kit
Select the matching RT² PreAMP Primer Mix for your PCR Array
Select your PCR
Arrays and RT² SYBR Green qPCR Master Mix
Important Note: Each RT² PreAMP
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 PreAMP reagents in this kit have been optimized to maximize the
sensitivity of our RT² Profiler PCR Arrays.
PreAMP Performance with formalin-fixed samples
Figure 1: Increased Positive Call Rate for Genes
Extracted from FFPE Samples
A. RNA extracted from a 5 year old Human FFPE spleen sample were
converted to cDNA with (gray bars) or without (red bars) pre-amplification
using RT² PreAMP PCR Master Mix and RT² PreAMP Primer Mix (Human Cancer
PathwayFinder). The unamplified and PreAMP amplified samples were
analyzed on the Human Cancer PathwayFinder RT² Profiler PCR Arrays
(PAHS-033), which contains 84 pathway-specific assays, plus controls,
including 5 assays for housekeeping genes. The threshold cycle values (Ct)
were obtained. Any genes with a Ct<35 were considered to be present.
B: Identify Genes Previously "Absent" with
RNA extracted from a Human FFPE spleen sample were converted to cDNA with
(gray bars) or without (red bars) pre-amplification with RT² PreAMP
Mix and RT² PreAMP Primer Mix (Human Cancer PathwayFinder). The
unamplified and PreAMP amplified samples were analyzed on the Human
Cancer PathwayFinder RT² Profiler PCR Arrays (PAHS-033), and the threshold
cycle values (Ct) were obtained. Shown here are 14 genes that are called
"Absent" (Ct>35) in the unamplified sample while these genes are
shown to be detectable in the PreAMP sample.
Figure 2: Unbiased Amplification - Highly
comparable ΔΔCt values between PreAMP pre-amplified and unamplified
cDNA from RNA extracted from FFPE spleen and intestine samples.
A. First-strand cDNA was synthesized from the extracted RNA samples,
and 1/4 of the RT product was used for pre-amplification with RT² PreAMP PCR
Master Mix plus Human Cancer PathwayFinder RT² PreAMP Primer Mix.
Unamplified cDNA synthesized from the same spleen and intestine samples
were used as the control. PreAMP amplified and unamplified cDNA
samples were then analyzed on the Human Cancer PathwayFinder PCR Array,
with the threshold cycle (Ct) obtained. Only genes which have raw Cts lower
than 33 in both unamplified spleen and intestine samples are displayed (ΔΔCt
B. RNA extracted from a FFPE spleen sample were
converted to cDNA with or without pre-amplification with RT² PreAmp
Mix and RT² PreAMP Primer Mix (Custom). The unamplified and PreAMP
amplified samples were analyzed on a custom PCR array containing 45 genes.
Shown here is raw Ct comparison between unamplified and amplified cDNA.
Only genes which have Cts lower than 33 in the unamplified cDNA are shown
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