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FFPE RNA Extraction Kit & PreAMPlification


Enabling the Analysis of FFPE Samples using qRT-PCR
  • A streamlined and real-time PCR-based process for pathway/disease-specific gene expression analysis in archived formalin-fixed paraffin-embedded (FFPE) samples.
  • The combination of a simplified Xylene-free RNA extraction and a high-fidelity amplification process which maximizes sample recovery and mRNA detection.

Benefits

  • High-quality and high-yield total RNA isolation from FFPE samples in 70 minutes
  • Pre-AMP protocol significantly enhances qRT-PCR detection sensitivity
  • Simple procedure and robust performance

The scientists at SABiosciences have developed an innovative solution enabling the accurate qRT-PCR analysis of Formalin-fixed Paraffin-embedded (FFPE) samples. Together with RT²  Profiler PCR Arrays, you can now easily and reliably analyze gene expression profiles associated with a biological pathway or a diseased state from FFPE samples. Learn more

User Manual

 
Product List How It Works Kit Contents Reagents & Software
 
How to Order

Product Name  Catalog  #
RNeasy FFPE Kit 73504
RT² PreAMP cDNA Synthesis Kit 330451
RT² PreAMP Primer Mixes for ALL Pathway-Focused PCR Arrays Click here
All Pathway-Focused PCR Arrays Find Yours Here
Instrument-Specific Master Mixes Find Yours Here

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Product List How It Works Kit Contents Reagents & Software
 

RT² FFPE RNA Extraction Kit

  • Simplified RNA Isolation: No Xylene needed for the de-paraffinization step.
  • Improved RNA Recovery: More effectively reverses formalin cross-links than other methods.
  • Greater Real-Time PCR Sensitivity: Lowers real-time PCR threshold cycle values.

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:

  1. Order RNeasy FFPE Kit and the RT² PreAMP cDNA Synthesis Kit

  2. Select the matching RT² PreAMP Primer Mix for your PCR Array

  3. 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 PreAMP.
RNA extracted from a Human FFPE spleen sample were converted to cDNA with (gray bars) or without (red bars) pre-amplification with 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), 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 values shown).

B. RNA extracted from a FFPE spleen sample were converted to cDNA with or without pre-amplification with RT² PreAmp PCR Master 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 here.

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Product List How It Works Kit Contents Reagents & Software
 

1. RT² PreAMP cDNA Synthesis kit (Catalog # 330451)

  1. 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
  2.  PreAMP Components (enough for 48 Pre-amplification reactions)
    • One (1) tube of PA-030 (RT² PreAMP PCR Master Mix) 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 Primer Mix for a specific PCR Array and perform multiplex PCR for 8 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.

3. RT² PreAMP Primer Mix (Catalogue #: PFX-####)
RT² PreAMP Primer Mix contains a ready-to-use primer mix for amplifying pathway-specific cDNA templates prior to real-time PCR Array

  • One (1) tube of primer mix for 12 reactions

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Product List How It Works Kit Contents Reagents & Software
 

The complete RT² Profiler™ PCR Array System insures that you will get good results from your pathway-focused gene expression analysis experiment the first time, and every time guaranteed.

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, 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

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For more information on QIAGEN’s complete portfolio of molecular analysis of FFPE samples, click here.