QuantiTect Whole Transcriptome Kit

For unlimited real-time PCR analysis from precious RNA samples

Features

  • 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
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QuantiTect Whole Transcriptome Kit (100)

Cat. No. / ID: 207045

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
$3,375.00
Reactions
100
25
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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.

Product Details

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.

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.
See figures

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.

See figures

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.
See figures

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).

Supporting data and figures

Specifications

FeaturesSpecifications
AmplificationWhole total RNA
Starting materialPurified total RNA
Reaction time5 hours or 11 hours
ApplicationsReal-time PCR, gene expression analysis
Minimal pipetting volume needed1 µl
Samples per run (throughput)Mid
TechnologyReverse transcription followed by ligation and multiple displacement amplification (MDA)
Yield10 µg or 40 µg cDNA
Maximum input volume5 µl total RNA
Starting amount of total RNA>10 ng purified total RNA
Reaction volume50 µl

Resources

Kit Handbooks (1)
For preparation of cDNA from total RNA by whole transcriptome amplification
Supplementary Protocols (1)
Brochures & Guides (1)

FAQ

What is the maximum amount of RNA that can be used for amplification with the QuantiTect Whole Transcriptome Kit?

Up to 300 ng RNA can be used for amplification with the QuantiTect Whole Transcriptome Kit without any change in transcript representation.

 

 

FAQ ID -1591
Can cDNA prepared by any method be used as starting material in the ligation reaction of the QuantiTect Whole Transcriptome Kit?

No, that is not possible. The reagents used in the ligation reaction of the QuantiTect Whole Transcriptome protocol are only compatible with cDNA prepared using the QuantiTect Whole Transcriptome Kit.

 

 

FAQ ID -1589
Can degraded RNA be used with the QuantiTect Whole Transcriptome Kit?

RNA transcripts should be longer than 500 nucleotides for use with the QuantiTect Whole Transcriptome Kit. RNA quality needs to be tested for each sample individually.

 

 

 

FAQ ID -1586
Can less than 10 ng RNA be used with the QuantiTect Whole Transcriptome Kit?

Yes, less than 10 ng of RNA can be used with the QuantiTect Whole Transcriptome Kit if the RNA is of high quality and the abundance of the transcript of interest is high. Please see Appendix A in the QuantiTect Whole Transcriptome Handbook for details on the limitations for RNA amplification.

(Note that 1 ng of RNA corresponds to approximately 50 cells).

 

FAQ ID -1590
How long is cDNA generated with the QuantiTect Whole Transcriptome Kit?

cDNA amplified with the QuantiTect Whole Transcriptome Kit is 20 kb in length.

However, note that the amplified cDNA contains concatemers of transcript sequences, and does not correspond to full-length RNA transcripts.

 

FAQ ID -1613
Can the QuantiTect Whole Transcriptome reaction be stopped after the reverse transcription step to proceed with the ligation and amplification steps at a later time?
Yes, this is possible. The reverse transcription reaction can be stored at -20°C.  We recommend not to store the ligation reaction longer than overnight at 4°C before proceeding to the next step in the protocol.

For convenience, all incubation steps of the QuantiTect Whole Transcriptome protocol can be pre-programmed on a thermal cycler.
FAQ ID -1618
Is the QuantiTect Whole Transcriptome Kit suitable for miRNA amplification?

No, as miRNA is shorter than 500 nt, the QuantiTect Whole Transcriptome Kit is not suitable for the amplification of miRNA.

 

 

FAQ ID -1609
Can different incubation times be used for the 2 hour amplification step in the QuantiTect Whole Transcriptome protocol?

Minimum amplification time in the QuantiTect Whole Transcriptome procedure is 2 hours. Any amplification time between 2 and 8 hours can be used. cDNA yield will depend on the length of the amplification time.

 

 

-8
Can RNA purified from formalin-fixed, paraffin-embedded (FFPE) tissue be used with the QuantiTect Whole Transcriptome Kit?

Suitability of RNA templates for use with the QuantiTect Whole Transcriptome Kit depends on RNA quality and needs to be tested for each sample individually.

Ideally, RNA transcripts should be longer than 500 nucleotides. Although most FFPE samples provide enough RNA, the RNA is of insufficient quality.

 

FAQ ID -1583
What is the maximum volume of RNA in solution that can be used with the QuantiTect Whole Transcriptome Kit?

A maximum of 5 µl RNA eluate from RNeasy extraction procedures can be added to the reverse-transcription reaction with the QuantiTect Whole Transcriptome Kit.

 

 

FAQ ID -1616
Can amplification with the QuantiTect Whole Transcriptome Kit be extended to more than 8 hours to improve cDNA yields?

We do not recommend extending the amplification time when using the QuantiTect Whole Transcriptome Kit. Maximum cDNA yields are achieved after 8 hours. In addition, transcript representation may by affected by amplification times longer than 8 hours.

 

 

FAQ ID -1608
Why is there DNA in the no-template (negative) control when using the QuantiTect Whole Transcriptome Kit?

In no-template control reactions for the QuantiTect Whole Transcriptome Kit, primer–dimers can form. The highly processive Phi29 DNA polymerase used in the amplification step of the QuantiTect Whole Transcriptome procedure will extend these primer–dimers, leading to nonspecific amplification products.

These nonspecific products do not influence subsequent real-time PCR.

 

FAQ ID -1620
Is cDNA generated with the QuantiTect Whole Transcriptome Kit suitable for use in microarray analysis?

cDNA generated with the QuantiTect Whole Transcriptome Kit is not compatible with Affymetrix microarrays. We have not tested compatibility with self-spotted cDNA arrays.

 

 

 

 

 

FAQ ID -1587
How long can I archive cDNA generated with the QuantiTect Whole Transcriptome Kit?

The cDNA generated with the QuantiTect Whole Transcriptome Kit is as stable as purified DNA and can be stored for several years.

 

 

FAQ ID -1623
Which real-time PCR kits are recommended downstream of the QuantiTect Whole Transcriptome Kit?

We highly recommend any QuantiTect or QuantiFast Kit for quantitative PCR on cDNA generated with the QuantiTect Whole Transcriptome Kit.

 

FAQ ID -1592
Can cDNA prepared with the QuantiTect Whole Transcriptome Kit be reampliied using REPLI-g Kits?

We recommend amplification of the original starting material with the QuantiTect Whole Transcriptome Kit. If the original starting material is no longer available, the cDNA can be reamplified at least once using REPLI-g Kits.

We have not validated more than one round of reamplification.

 

FAQ ID -1611
Have you observed co-amplification of genomic DNA from RNA templates used in the QuantiTect Whole Transcriptome Procedure?

No, we have never observed coamplification of genomic DNA from RNA templates used in the QuantiTect Whole Transcriptome protocol when using RNA purified with RNeasy Kits without on-column DNase digestion.

 

 

FAQ ID -1619
Is it necessary to clean up cDNA prepared with the QuantiTect Whole Transcriptome Kit?

It is not necessary to clean up cDNA prepared with the QuantiTect Whole Transcriptome Kit if using it for real-time PCR. See the QuantiTect Whole Transcriptome Handbook for recommendations on cDNA dilution.

 

FAQ ID -1610
Does carrier RNA interfere with the QuantiTect Whole Transcriptome amplification?

Small amounts of carrier RNA, such as used with RNeasy Micro Kit, do not interfere.  However, larger amounts such as used with QIAamp MinElute Virus Kits, the QIAamp Viral RNA Mini Kit, or the QIAsymphony DSP Virus/Pathogen Kits, will interfere with the Whole Transcriptome amplification.

FAQ ID -3080
Does the QuantiTect Whole Transcriptome Kit work with RNA purified from bacteria, yeast, or plants?

Yes, the QuantiTect Whole Transcriptome Kit works with all RNA with a length of >500 nt.

 

 

FAQ ID -1615
What primers are used in the reverse-transcription step of the QuantiTect Whole Transcriptome procedure?

Random primers as well as oligo-dT primers are used in the reverse-transcription step of the QuantiTect Whole Transcriptome protocol.

The QuantiTect Whole Transcriptome Kit amplifies cDNA derived from all regions of RNA transcripts, including 5' ends, but does not provide amplified cDNA corresponding to full-length RNA transcripts.

 

FAQ ID -1584
Can T-Script® enzyme of the QuantiTect Whole Transcriptome Kit be substituted by Quantiscript Reverse Transcriptase?

No, the T-Script® enzyme of the QuantiTect Whole Transcriptome Kit is an optimized blend for whole transcriptome amplification and cannot be substituted by Quantiscript Reverse Transcriptase of the QuantiTect Reverse Transcription Kit, or any other reverse-transcription enzyme.

 

FAQ ID -1617
Is it necessary to include a genomic DNA removal step with the QuantiTect Whole Transcriptome Kit?

Our experiments do not show any need to include a step for genomic DNA removal with the QuantiTect Whole Transcriptome Kit.

 

FAQ ID -1614
Does cDNA amplified with the QuantiTect Whole Transcriptome Kit correspond to full-length RNA transcripts?

The QuantiTect Whole Transcriptome Kit amplifies cDNA from all regions of RNA transcripts, including 5' ends, but does not generate amplified cDNA corresponding to full-length RNA transcripts.

 

 

 

FAQ ID -1585
Can the QuantiTect Whole Transcriptome Kit be used for amplification of RNA from LCM samples?

Yes, use the RNeasy Micro Kit or RNeasy Mini Kit to purify RNA from laser-capture microdissected (LCM) samples prior to amplification with the QuantiTect Whole Transcriptome Kit. Note, however, that carrier RNA has to be avoided in the RNA purification procedure as it may affect specific amplification of transcript sequences.

RNeasy Kits ensure that the RNA is of sufficient quality. Degraded RNA cannot be used for amplification.

 

FAQ ID -1612