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RT2 PreAMP Pathway Primer Mixes

For amplification of cDNA templates prior to analysis on RT² Profiler PCR Arrays
  • Primer mixes for human, mouse, and rat
  • Minimal hands-on time
  • Robust performance on small samples

RT² PreAMP cDNA Synthesis Kit and RT² PreAMP Pathway Primer Mixes are a novel technology enabling gene expression analysis using as little as 1 ng total RNA. A  proprietary amplification process increases the amount of cDNA for subsequent PCR array analysis. Starting sample types include fine needle biopsy and laser captured microdissection samples, stem cell clusters or embryoid bodies, and fluorescence-activated cell sorter (FACS) generated cell populations. Primer mixes are available for all cataloged human, mouse, and rat RT2 Profiler PCR Arrays.

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The RT2 PreAMP Pathway Primer Mixes is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Increased positive call rate.
First strand cDNA was synthesized from 40 ng mouse total RNA with (light blue bars) or without (dark blue bars) preamplification with RT² PreAMP PCR Mastermix and RT² PreAMP Pathway Primer Mix. The unamplified and amplified samples were then analyzed on the Mouse Inflammatory Cytokines & Receptors RT² Profiler PCR Array and the threshold CT were obtained. Genes with CT ≥35 were considered to be "absent". In the unamplified sample, 75% of the genes analyzed were present. The positive call rate was increased to 100% in the amplified sample. The 22 genes shown were called "absent" in the unamplified sample and were detectable in the preamplified sample.
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Simple template amplification and gene-expression analysis.
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Unbiased amplification process.
First strand cDNA was synthesized from 5 ng of human liver tumor RNA. One-quarter of the first strand cDNA was then used for preamplification with RT² PreAMP PCR Master Mix plus Human Cancer PathwayFinder RT² PreAMP Pathway Primer Mix. Unamplified cDNA synthesized from 500 ng of the same sample was used as a control. Preamplified and unamplified cDNA samples were then analyzed on the Human Cancer PathwayFinder RT² Profiler PCR Array and the DCT values were obtained. Concordance between preamplified and unamplified samples was evaluated by regression analysis. Data points with CT ≥35 were considered to be “absent” genes and were excluded from analysis. The lines show a linear regression fit with the R2 and slope indicated in the graph.
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Faithfully amplified biology.
First strand cDNA was synthesized from 1 ng human liver tumor RNA or universal RNA. One-quarter of the first strand cDNA was then used for preamplification with RT² PreAMP PCR Master Mix plus Human Cancer PathwayFinder RT² PreAMP Pathway Primer Mix. Unamplified cDNA synthesized from 500 ng of each corresponding RNA sample was used as a control. Preamplified and unamplified cDNA samples were analyzed on the Human Cancer PathwayFinder RT² Profiler PCR Array and the fold change in gene expression between liver tumor and universal RNA for each gene was obtained using the ΔΔCT method.
Performance
Increased positive call rate and unbiased amplification for unbiased results

More genes are detected after preamplification using the RT² PreAMP Pathway Primer Mix (see figure "Increased positive call rate").

The amplification process is unbiased, resulting in highly comparable ΔCT values between preamplified and unamplified cDNA, as evaluated by regression analysis (see figure "Unbiased amplification process").

There is a high correlation of gene expression fold change results between preamplified and unamplified samples (see figure "Faithfully amplified biology").

Principle
RT² PreAMP technology utilizes multiplex tandem PCR to preamplify gene-specific cDNA with minimal bias. Following standard reverse transcription, an RT2 PreAMP Pathway Primer Mix is used to amplify templates and enable gene expression analysis on up to 4 different  RT2 Profiler PCR Arrays (see flowchart "Simple template amplification and gene-expression analysis").
Procedure

First strand cDNA is first synthesized from up to 12 different RNA samples using the RT2 PreAMP cDNA Synthesis Kit. The cDNA is then preamplified for a pathway-specific set of genes. Each first strand cDNA synthesis reaction can be amplified using up to 4 different RT2 PreAMP Pathway Primer Mixes, allowing gene expression analysis on up to 4 different RT2 Profiler PCR Arrays. The Side Reaction Reducer included in the RT2 PreAMP cDNA Synthesis Kit eliminates the residual primers from preamplification, enabling accurate detection on RT2 Profiler PCR Arrays. To complete the PCR array procedure, preamplified templates are mixed with an instrument-specific, ready-to-use RT² SYBR® Green Mastermix. 

Applications
cDNA templates generated using the RT2 PreAMP cDNA Synthesis Kit in conjunction with RT2 PreAMP Pathway Primer Mixes are ready for gene expression profiling using RT² Profiler PCR Arrays.

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Brochures & Guides (1)
Simultaneously profile mRNA, miRNA and lncRNA using a simple, complete workflow
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Kit Handbooks (1)
For synthesis and preamplification of cDNA from small RNA samples and RNA from formalin-fixed, paraffin-embedded samples
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