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QuantiTect Whole Transcriptome Kit

For unlimited real-time PCR analysis from precious RNA samples
  • High cDNA yields for unlimited qPCR analysis and archiving
  • Equal amplification of all cDNA and all transcript regions
  • Fast and easy protocol with optimized reagents

The QuantiTect Whole Transcriptome Kit enables the preamplification and reverse transcription of limited amounts of RNA to high yields of cDNA for unlimited gene expression analysis using real-time PCR. An optimized protocol ensures a fast and easy procedure. The kit consists of a complete set of enzymes and buffers for whole transcriptome amplification (WTA). Innovative modification of Phi 29 polymerase technology allows yields of up to 40 μg cDNA from as little as 1 ng RNA. The uniquely high processivity of this polymerase guarantees the generation of cDNA containing uniformly amplified targets to ensure reliable gene expression analysis in real-time PCR.

Cat No./ID: 207043
QuantiTect Whole Transcriptome Kit (25)
$1,722.00
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For 25 x 50 µl reactions: 25 µl T-Script Enzyme, 400 µl T-Script Buffer, 25 µl Ligation Enzyme 1, 25 µl Ligation Enzyme 2, 200 µl Ligation Reagent, 600 µl Ligation Buffer, 25 µl REPLI-g Midi DNA Polymerase, 730 µl REPLI-g Midi Reaction Buffer
Cat No./ID: 207045
QuantiTect Whole Transcriptome Kit (100)
$5,626.00
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For 100 x 50 µl reactions: 100 µl T-Script Enzyme, 400 µl T-Script Buffer, 100 µl Ligation Enzyme 1, 100 µl Ligation Enzyme 2, 200 µl Ligation Reagent, 600 µl Ligation Buffer, 100 µl REPLI-g Midi DNA Polymerase, 2 x 1.45 ml REPLI-g Midi Reaction Buffer
The QuantiTect Whole Transcriptome Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
0
Reproducible cDNA yields.
Total RNA (10 ng) from different blood samples was amplified for 2 or 8 hours using the QuantiTect Whole Transcriptome Kit. cDNA yields were determined using PicoGreen reagent.
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Equal amplification of 5' and 3' regions.
Total RNA (1 ng) was amplified using the QuantiTect Whole Transcriptome Kit. This was followed by real-time PCR using 10 ng cDNA, primers specific for β-actin, and the QuantiTect SYBR® Green PCR Kit. Amplicons corresponding to the 5' and 3' regions of the β-actin transcript were detected with similar CT values, indicating that the QuantiTect Whole Transcriptome Kit provided equal amplification of all transcript regions.
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Procedure.

cDNA is amplified from purified RNA with 3 sequential reactions: reverse transcription, ligation, and whole transcriptome amplification.

3
Reliable real-time PCR analysis.
Replicate RNA samples (10 ng each) were amplified using the QuantiTect Whole Transcriptome Kit. Real-time PCR of the indicated targets was then performed using 10 ng cDNA and the QuantiFast Probe PCR Kit on the Mx3005P system. The overlapping curves indicate highly reproducible whole transcriptome amplification. ACTB: β-actin; HPRT1: Hypoxanthine phosphoribosyltransferase 1.
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Whole transcriptome amplification.
cDNA is first synthesized from template RNA and then ligated (not shown). REPLI-g DNA polymerase moves along the cDNA template strand displacing the complementary strand. The displaced strand becomes a template for replication, allowing high yields of cDNA to be generated.
5
Preservation of transcript profile.
Total RNA (1 ng) was amplified using the QuantiTect Whole Transcriptome Kit for 2 hours (WTA 2 h) or 8 hours (WTA 8 h), followed by real-time PCR of 5 targets using 10 ng cDNA. As a control, RNA was reverse transcribed without amplification using the Sensiscript RT Kit (Non-WTA), and 1/5 of the RT reaction was used in real-time PCR. Real-time PCR was carried out using the QuantiTect Probe PCR Kit. [A] Relative gene expression levels of 5 transcripts (normalization to HPRT1) are similar in the WTA 2 h, WTA 8 h, and Non-WTA samples, as shown by the overlapping points. [B] A plot of ΔCT values (CT value for each target minus CT value for internal reference β-actin) for WTA 8 h samples (Y-axis) against those for Non-WTA samples (X-axis) is highly linear. The data in [A] and [B] demonstrate that the QuantiTect Whole Transcriptome Kit preserves the transcript profile. TP53: Tumor protein p53; NFKB1: nuclear factor of kappa light polypeptide gene enhancer in B-cells 1; HPRT1: hypoxanthine phosphoribosyltransferase 1; TBP: TATA box binding protein; TNFRSF6B: tumor necrosis factor receptor superfamily, member 6b, decoy.
Performance

The QuantiTect Whole Transcriptome Kit provides highly uniform amplification of all transcripts, which is essential for reliable gene expression analysis. All mRNA transcripts are amplified with equal representation at both 5' and 3' regions (see figure "Equal amplification of 5' and 3' regions"). Preservation of the transcript profile is demonstrated in the figure "Preservation of transcript profile", where WTA-amplified cDNA prepared using the QuantiTect Whole Transcriptome Kit is compared with nonamplified cDNA prepared using a reverse transcriptase.

High and reproducible cDNA yields of up to 40 μg can be achieved (see figure "Reproducible cDNA yields"). This allows unlimited real-time PCR analysis with highly reproducible CT values (see table and figure "Reliable real-time PCR analysis"). In addition, since cDNA is more stable than RNA, it can be safely archived for future studies.

High yields of cDNA for real-time PCR analysis
Starting materialAmplification time Typical yield of amplified productNumber of real-time PCR analyses*
10 ng RNA 2 hours Up to 10 µg cDNA 1000
10 ng RNA 8 hours Up to 40 µg cDNA 4000
* Number of real-time PCR analyses possible using cDNA from a single QuantiTect whole transcriptome amplification (WTA) reaction. Without WTA, only 1 reliable real-time RT-PCR analysis is possible from 10 ng RNA.
Principle

Gene expression profiling can be limited by the small amount of biological sample available. By using the QuantiTect Whole Transcriptome Kit, unlimited real-time PCR analyses of small and precious samples are possible. The QuantiTect Whole Transcriptome Kit is optimized for whole transcriptome amplification from as little as 1 ng RNA, or RNA corresponding to about 50 cells. Even lower amounts of RNA can be used, depending on the quality of the RNA and the abundance of the transcript of interest.

The QuantiTect Whole Transcriptome Kit contains reverse transcriptase, DNA polymerase, and optimized buffers and reagents for the amplification of all transcripts within an RNA sample. The kit integrates cDNA synthesis with the proven quality of REPLI-g technology for nonbiased sequence amplification. REPLI-g amplification uses Multiple Displacement Amplification (MDA) technology, which carries out isothermal sequence amplification using a uniquely processive DNA polymerase (see figure "Whole transcriptome amplification"). This technology ensures unbiased amplification of all transcript regions, even of 5' ends, providing unlimited cDNA highly suited for real-time PCR.

Procedure
Unlimited cDNA representing the entire transcriptome is prepared from total RNA in a simple 3-step protocol (see flowchart "Procedure"). RNA is first reverse transcribed into cDNA using an optimized reverse transcription mix containing T-Script Enzyme with random and oligo-dT primers. The synthesized cDNA is ligated using a high-efficiency ligation mix, and then amplified using an amplification mix based on proven REPLI-g technology.
Applications

cDNA prepared using the QuantiTect Whole Transcriptome Kit is intended for use in real-time PCR analysis with QuantiFast or Rotor-Gene Kits and can be archived for future analysis. The cDNA is not suitable for use in microarray analysis. For whole genome amplification from small or precious samples, QIAGEN offers REPLI-g kits and REPLI-g Service. Amplification is highly uniform with minimal sequence bias, providing DNA suitable for applications such as genotyping and Comparative Genome Hybridization (CGH).

Features
Specifications
Amplification Whole total RNA
Applications Real-time PCR, gene expression analysis
Maximum input volume 5 µl total RNA
Minimal pipetting volume needed 1 µl
Reaction time 5 hours or 11 hours
Reaction volume 50 µl
Samples per run; throughput Mid
Starting amount of total RNA >10 ng purified total RNA
Starting material Purified total RNA
Technology Reverse transcription followed by ligation and multiple displacement amplification (MDA)
Yield 10 µg or 40 µg cDNA

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Kit Handbooks (2)
For preparation of cDNA from total RNA by whole transcriptome amplification
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全トランスクリプトーム増幅によりトータルRNAからcDNAを調製
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