QuantiTect Whole Transcriptome Kit

For unlimited real-time PCR analysis from precious RNA samples

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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
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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.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

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

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
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)
Gene Expression Analysis (1)
Certificates of Analysis (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
How do I setup and validate a multiplex PCR assay with QIAGEN PCR kits?

Ensure PCR amplicons are as short as possible, ideally 60–150 bp. Always use the same algorithm or software to design the primers and probes. For optimal results, only combine assays that have been designed using the same parameters.

 

Check the functionality of each set of primers and probes in individual assays before combining the different sets in the multiplex assay. Choose compatible reporters and quenchers based on a specific instrument. See How do I select appropriate reporter and quencher combinations for multiplex PCR.

 

FAQ ID -9093
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 I use uracil-N-glycosylase (UNG) with the QuantiFast and Rotor-Gene PCR kits?

No. UNG treatment does not provide any advantage for the QuantiFast and Rotor-Gene PCR kits because the mastermixes do not contain dUTP. Use the QuantiTect kits if you intend to use the UNG treatment.

FAQ ID -9092
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
Why do I see multiple high-intensity peaks in my qPCR dissociation curve at temperatures less than 70ºC?

If the extra peaks seem irregular or noisy, do not occur in all samples, and occur at temperatures less than 70 ºC, then these peaks may not represent real PCR products and instead may represent artifacts caused by instrument settings.

 

Usually extra peaks caused by secondary products are smooth and regular, occur reproducibly in most samples, and occur at temperatures greater than 70 ºC. Characterization of the product by agarose gel electrophoresis is the best way to distinguish between these cases. If only one band appears by agarose gel then the extra peaks in the dissociation curve are instrument artifacts and not real products. If this is the case, refer to the thermal cycler user manual, and confirm that all instrument settings (smooth factor, etc.) are set to their optimal values.

 

FAQ ID -90990
How do I quantify gene expression levels if the amplification efficiencies are different between the genes of interest and endogenous reference gene?

The REST 2009 (Relative Expression Software Tool) software applies mathematic models that compensate for the different PCR efficiencies of the gene of interest and reference genes. In addition, the software can use multiple reference gene normalization to improve the reliability of result, as well as provides statistical information suitable for robust comparison of expression in groups of treated and untreated. QIAGEN offers the REST 2009 software free of charge.

FAQ ID -9095
What do I do if no fluorescent signal is detected in a real-time PCR assay?

Check the template quality and integrity by amplifying an endogenous control gene. Check the amplicon by QIAxcel Advanced system or agarose gel electrophoresis to show that amplification was successful.

 

Determine whether the gene of interest is expressed in your sample. See How can I find out if my gene of interest is express in a specific tissue type or cell line.  Ensure the assay setup and cycling conditions are correct, and that the data collection channel matches the emission wavelength of the fluorescent dye used. Use a control sample in which the gene of interest is definitely expressed.

 

If the issue persists, please send the original run file to QIAGEN Technical Services.

FAQ ID -9091
How do I select appropriate reporter and quencher combinations for multiplex PCR?

For duplex analysis, using non-fluorescent quenchers (e.g., Black Hole Quencher®) is preferred over fluorescent quenchers (e.g., TAMRA fluorescent dye). For triplex and 4-plex analysis, QIAGEN strongly recommends using non-fluorescent quenchers. Generally, use the green channel, the yellow channel, and the orange and crimson channels to detect the least abundant target, the second least abundant target, and the two most abundant targets, respectively. For instrument-specific recommendations, please see the handbooks for the QuantiTect Multiplex PCR kit, QuantiFast Multiplex kit or Rotor-Gene Multiplex kit.

 

FAQ ID -9094
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
Can I skip the gDNA wipeout buffer treatment step for the QuantiTect Reverse Transcription Kit?

The gDNA wipeout buffer incubation step can be skipped when the total RNA is free from genomic DNA. However, the gDNA wipeout buffer is still required to be added because the reverse transcription step is optimized in the presence of components in the gDNA wipeout buffer.

FAQ ID -9098
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
How should I handle and store absolute quantitation standards for real-time experiments?
Store the standards at a high concentration in aliquots at -20oC to -70oC. If using low concentrations, stabilize standards with carrier nucleic acid. It is always best to use freshly diluted standards for each experiment. If possible, use siliconized tubes for standard (and target) dilutions. This will prevent any unspecific binding of nucleic acids to the plastic.
FAQ ID -9099
How do I ensure reliable results for High Resolution Melting (HRM) assays?

Reliable HRM analysis results depend on template quality, highly specific HRM PCR kit with a saturation dye, a real-time instrument with HRM capability, and powerful software package. Factors critical for successful HRM analysis are:

 

  • Use the same genomic DNA purification procedure for all samples being analyzed by HRM. This avoids variation due to differing composition of elution buffers.
  • DNA template concentrations should be normalized using the same dilution buffer. Ensure the CT values are below 30 and differ no more than 3 CT values across individual samples.
  • Design assays with amplicon length 70–350 bp. For SNP analysis, use amplicon length 70–150 bp.
  • Always start with 0.7 µM primer concentration

 

For more details, please refer to the HRM Technology – FAQs and the Critical Success Factor for HRM performance.

FAQ ID -9097
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
How do I avoid collecting a fluorescence reading from primer-dimer with the QuantiTect SYBR Green PCR Kit?

Depending on primer design and copy number of target, primer-dimer may occur and its signal might be detected. Typical strategies against this are to optimize PCR conditions and/or redesign the assay.

 

Alternatively, an additional data-acquisition step can be added to the 3-step cycling protocol. First, determine the melting temperatures (Tm) for both the amplicon and the primer-dimer. Then, add a 15 second data-acquisition step with a temperature that is higher than the primer-dimer Tm, but approximately 3ºC lower than the specific amplicon Tm.

FAQ ID -9096
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