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RT² PreAMP cDNA Synthesis Kit and Primer Mixes


Enabling the Analysis of 1 ng Total RNA on RT² Profiler PCR Arrays

RT² PreAMP cDNA Synthesis Kit and Primer Mixes are a breakthrough technology enabling the gene expression analysis using as little as 1 ng total RNA. It employs a proprietary amplification process to faithfully increase the amount of respective cDNA for subsequent PCR Array analysis. This technology empowers PCR Array to accurately analyze nanogram level of total RNA. This kit is intended for synthesis followed by pre-amplification of first strand cDNA from limited amount of total RNA samples for gene expression analysis with our RT² Profiler™ PCR Arrays. The samples that are enabled for real-time PCR Arrays include:
  1. Fine needle biopsy (FNB) samples.
  2. Laser captured microdissection (LCM) samples.
  3. Stem cell clusters or embryoid bodies.
  4. Fluorescence-Activated Cell Sorter (FACS) generated cells.

Learn more about the complete PCR Array System.

User Manual

 
Product List How It Works Kit Content Reagents & Software
 
Product Name  Species  Catalog  #
RT² PreAMP cDNA Synthesis Kit    330451
RT² PreAMP cDNA Synthesis Primer Mixes for Popular Pathway-Focused PCR Arrays
Cancer PathwayFinder  Human PBH-033Z
Apoptosis  Human PBH-012Z
Angiogenesis Human PBH-024Z
Mesenchymal Stem Cells  Human PBH-082Z
Embryonic Stem Cells  Human PBH-081Z
Extracellular Matrix and Adhesion Molecules  Human PBH-013Z
Inflammatory Cytokines and Receptors  Human PBH-011Z
Mouse PBM-011Z
Toll-like Receptor Signaling Pathway Human PBH-018Z
Mouse PBM-018Z
For RT² PreAMP cDNA Synthesis Primer Mixes for all other Pathway-Focused PCR Arrays, please click here

Benefits of RT² PreAMP cDNA Synthesis Kit:

  • Robust Performance on Small Samples: 1 ng total RNA can be used for 4 different PCR Arrays
  • Easy Workflow and Designed for Routine Use: Simple and quick procedures with minimal hands-on time to pre-amplify target templates under two hours
  • Superior Sensitivity: Maximally enhances the sensitivity of RT² Profiler PCR Array to analyze limited amounts of cDNA
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Product List How It Works Kit Content Reagents & Software
 

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 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:

  • 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 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:

  1. Select your PCR Arrays and RT² SYBR Green qPCR Master Mix

  2. Order RT² PreAMP cDNA Synthesis Kit

  3. Select the matching RT² PreAMP cDNA Synthesis Primer Mix for your PCR Array

Important Note: Each RT² 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 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 (grey bars) or without (red bars) pre-amplification with RT² PreAMP Master Mix and RT² 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 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 ΔCt 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 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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Product List How It Works Kit Content Reagents & Software
 

1. RT² PreAMP cDNA Synthesis Kit (Catalogue # 330451)

A: RT² First Strand cDNA Synthesis Components (enough for 12 20-µl RT reactions) -

  • One (1) tube of GE (5X gDNA Elimination Buffer)
  • One (1) tube of BC3 (5X Reverse Transcription Buffer 3)
  • One (1) tube of RE (cDNA Synthesis Enzyme Mix)
  • One (1) tube of RI (RNase Inhibitor)
  • One (1) tube of P2 (Primer and External Control Mix)
  • One (1) tube of RNase-free H2O

B: PreAMP Components (enough for 48 Pre-amplification reactions)

  • One (1) tube of RT² PreAMP PCR Mastermix containing 600µl of 2X solution and enough for 4 25-µl standard reactions for each of the 12 cDNA synthesized (48 pre-amplification reactions)
  • One (1) tube of SR1 (Side Reaction Reducer ) containing 96µl SR1 enough for 4 standard reactions for each of 12 samples (48 pre-amplification reactions)

The RT² PreAMP cDNA Synthesis Kit contains all the reagents required for the reverse transcription of RNA. The RT² PreAMP PCR Master Mix (2X) is required for the multiplex PCR-based pre-amplification and contains PCR buffer formulated for multiplex PCR, a high-performance HotStart DNA Taq polymerase and nucleotides. The Side Reaction Reducer that comes with the kit eliminates any residual PreAMP primers. After performing the reverse transcription step, simply add the master mix to PCR tubes along with your cDNA template and the RT² PreAMP cDNA Primer Mix for a specific PCR Array and perform multiplex PCR for 12 cycles on any end-point thermal cycler. Then treat the amplified samples with the Side Reaction Reducer, and the samples will be ready for use in the RT² Profiler PCR Arrays. The cDNA product from each first strand synthesis reaction is sufficient for performing the PreAMP step for four different PCR Arrays.

2. RT² PreAMP cDNA Synthesis Primer Mix
RT² PreAMP cDNA Primer Mix contains ready-to-use primer mix for amplifying pathway-specific cDNA templates prior to real-time PCR Array

  • One (1) tube of primer mix

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Product List How It Works Kit Content Reagents & Software
 

The Complete RT² Profiler™ PCR Array System Includes:

Our convenient and high-quality real-time RT-PCR accessory products complete the RT² Profiler™ PCR Array System and the RT² PCR Primer Assays. The other components needed for real-time PCR analysis are SYBR Green PCR master mix, first strand synthesis kit, and an optional RNA QC array.

Other recommended accessory products

NEW: RT² RNA QC PCR Array
Tests for RNA Quality and Inhibitors of RT-PCR Analyses, optional

NEW: RT² qPCR-Grade RNA Isolation Kit
From the experts in isolating RNA free of genomic DNA for real-time RT-PCR

QIAGEN RNeasy FFPE Kit
Optimized isolation of total RNA from FFPE blocks and slides< for real-time RT-PCR

 

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