QuantiTect Whole Transcriptome Kit

对珍贵的RNA样本进行无限次real-time PCR分析

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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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100

QuantiTect Whole Transcriptome Kit 旨在用于分子生物学应用。该产品不能用于疾病诊断、预防和治疗。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

高cDNA产量用于无限次分析和长期保存
均一扩增所有cDNA和所有转录本区段
经优化的试剂，快速简单的操作流程

Product Details

QuantiTect Whole Transcriptome Kit对有限的RNA样品进行预扩增和逆转录，获得高产量cDNA，用于无限次real-time PCR基因表达分析。该试剂盒包含全转录扩增所需的全部酶和缓冲液。对Phi29聚合酶创新的修饰确保从低至1 ng RNA中制备多至40 μg cDNA。该聚合酶独特的加工过程可制备高度统一的cDNA，确保通过real-time PCR进行可靠的基因表达分析。

Performance

QuantiTect Whole Transcriptome Kit确保所有转录子高度一致的扩增，这对于基因表达分析是必需的。所有mRNA转录子以相同的形式同时在5'和3'末端扩增（参见" Equal amplification of 5' and 3' regions"）。图" Preservation of transcript profile", 显示高度稳定的转录子，该图将分别应用QuantiTect Whole Transcriptome Kit全基因组扩增的cDNA和用逆转录酶制备的未扩增cDNA进行了对比。

可获得多至40 μg的可重复、高产量的cDNA（参见" Reproducible cDNA yields"）。能够进行无限次real-time PCR分析，并具有高度重复性的C_T值（参见" Reliable real-time PCR analysis"）。此外，由于cDNA比RNA更稳定，可以存档用于以后的研究工作。

高产量的cDNA可用于real-time PCR分析
起始材料	扩增时间	扩增产物的常规产量	可进行的real-time PCR次数*
10 ng RNA	2小时	至多10 µg cDNA	1000
10 ng RNA	8小时	至多40 µg cDNA	4000

See figures

Equal amplification of 5' and 3' regions.

Preservation of transcript profile.

Reproducible cDNA yields.

Reliable real-time PCR analysis.

Principle

获得的生物样本量太少可能限制基因表达谱分析。使用QuantiTect Whole Transcriptome Kit可对少量珍贵的样品进行无限次real-time PCR分析。QuantiTect Whole Transcriptome Kit经优化，用于1 ng RNA或相当于约50个细胞RNA的全转录扩增，甚至可以用更少量的RNA，这些取决于RNA的品质和转录子的丰度。

QuantiTect Whole Transcriptome Kit含有逆转录酶、DNA聚合酶和优化的缓冲液及试剂，可对一个RNA样本的所有转录本进行扩增。此试剂盒整合了经质量验证的REPLI-g技术，通过非偏向序列扩增来合成cDNA。REPLI-g扩增采用Multiple Displacement Amplification（MDA）技术，应用独特加工的DNA聚合酶实现等温扩增（参见" Whole transcriptome amplification"）。该技术确保包括5’末端在内的所有转录区的同等扩增，从而获得可用于real-time PCR的足够多的cDNA。

See figures

Whole transcriptome amplification.

Procedure

从总RNA开始经过3个步骤可获得大量代表了整个转录子的cDNA（参见""）。首先，应用优化的含有T-Script Enzyme的逆转录混合物（含有随机引物和oligo-dT引物）将RNA转录为cDNA。用高效的连接混合物连接，然后用基于经验证的REPLI-g技术的扩增混合物扩增cDNA。

See figures

Procedure.

Applications

QuantiTect Whole Transcriptome Kit制备的cDNA可用于QuantiFast或Rotor-Gene Kits进行的real-time PCR分析，且可以储存用于以后的分析，但不适用于微阵列分析。QIAGEN提供REPLI-g Kits and Service，用于小量或珍贵样品的全基因组扩增。扩增高度一致，具有最小的序列偏好，可制备适合于基因分型和Comparative Genome Hybridization等应用的DNA。

Supporting data and figures

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.

Specifications

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

Resources

For preparation of cDNA from total RNA by whole transcriptome amplification

Download Safety Data Sheets for QIAGEN product components.

For preparation of cDNA from total RNA by whole transcriptome amplification

FAQ

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

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

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

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

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

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

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

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

FAQ ID -1609

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

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

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

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

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

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

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

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

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

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

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

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

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

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

FAQ ID -1592

Store the standards at a high concentration in aliquots at -20^oC to -70^oC. 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

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 C_T values are below 30 and differ no more than 3 C_T 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

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

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

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

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

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

FAQ ID -1615

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

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

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 (T_m) 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 T_m, but approximately 3ºC lower than the specific amplicon T_m.

FAQ ID -9096

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

FAQ ID -1614

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

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