QuantiTect Whole Transcriptome Kit

貴重なRNA サンプルから膨大な数のリアルタイムPCR 解析を実現

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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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QuantiTect Whole Transcriptome Kitは分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。

✓ オンライン注文による24時間年中無休の自動処理システム

✓ 知識豊富で専門的な製品&テクニカルサポート

✓ 迅速で信頼性の高い（再）注文

特徴

多数の定量PCR解析やアーカイブを実現する高いcDNA収量
cDNAおよび転写物の全領域から均一に増幅
至適化された試薬で迅速かつ簡単な実験操作

製品詳細

QuantiTect Whole Transcriptome Kit では、限られた量のRNAから増幅および逆転写反応により高収量のcDNAのを得ることができ、リアルタイムPCRによる多数の遺伝子発現解析が可能になります。至適化済みのプロトコールにより迅速かつ簡単に操作できます。本キットは、全トランスクリプトーム増幅（whole transcriptome amplification；WTA）のための酵素および試薬のセットです。Phi29 ポリメラーゼテクノロジーの画期的な改良により、わずか1 ng のRNAから最高40 μg のcDNAが得られます。このポリメラーゼの画期的な高い伸長性により、均一に増幅されたターゲットを含むcDNAが合成され、リアルタイムPCRで信頼性のある遺伝子発現解析を実現します。

パフォーマンス

QuantiTect Whole Transcriptome Kit による全転写物の均一な増幅は、信頼できる遺伝子発現解析において必須条件です。全てのmRNA転写物が5'領域および3'領域の両方で均一に増幅されます（図 " 5’領域および3’領域を均一に増幅"）。QuantiTect Whole Transcriptome Kit でWTA増幅したcDNA と、逆転写酵素で調製した増幅なしのcDNAを比較した結果を図 " 転写物プロファイルの保持"に示しています。これは、転写物プロファイルが保持されていることを証明しています。

最高40 μg までの高収量cDNAが再現性良く得られます（図 " 一定したcDNA収量"）。この特長によりリアルタイムPCR 解析を何回も行なうことが可能で、C_T 値も一定しています（表および図 " 信頼できるリアルタイムPCR解析"）。さらにcDNAはRNAよりも安定であるため、将来行なう研究実験用に、安全に保存することができます。

リアルタイムPCR用に高収量でcDNA を産生
スタートサンプル	増幅時間	増幅産物の一般的な収量	解析可能なリアルタイムPCR解析の回数*
10 ng RNA	2 時間	最高 10 µg/反応	1000
10 ng RNA	8 時間	最高 40 µg/反応	4000

図参照

Equal amplification of 5' and 3' regions.

Preservation of transcript profile.

Reproducible cDNA yields.

Reliable real-time PCR analysis.

原理

使用可能な生体サンプルが微量の場合、遺伝子発現プロファイル解析が制限されることがあります。QuantiTect Whole Transcriptome Kitを用いることで、微量かつ貴重なサンプルから膨大な数のリアルタイムPCR 解析が可能になります。QuantiTect Whole Transcriptome Kitは、わずか1 ng のRNA あるいは約50個の細胞に相当するRNAからの全トランスクリプトーム増幅用に至適化されています。RNAの品質および目的の転写物の量によっては、さらに少量のRNAでも使用できます。

QuantiTect Whole Transcriptome Kitには、RNAサンプル中の全転写物の増幅のための逆転写酵素、DNAポリメラーゼ、至適化済みバッファーと試薬が含まれています。本キットは、バイアスのない配列増幅のために、実証済みのREPLI-g テクノロジーを用いたcDNA合成を組み込んでいます。REPLI-g 増幅は、伸長性が高いDNAポリメラーゼを用いて等温でゲノム増幅ができるMultiple Displacement Amplification（MDA）テクノロジーを利用しています（図 " 全トランスクリプトーム増幅）。本テクノロジーは転写物の全領域（5'領域からでも）からバイアスのない均一な増幅を実現するので、リアルタイムPCRに最適な高収量のcDNAが得られます。

図参照

Whole transcriptome amplification.

操作手順

わずか3 ステップの操作手順でトータルRNAからトランスクリプトームの全体像を表すcDNAを高い収量で調製できます（フローチャート ""）。まずT-Script Enzyme およびrandom プライマーとoligo-dT プライマーを含む至適化済みの逆転写反応ミックスを用いてRNAをcDNAに逆転写します。合成したcDNAを効率の高いライゲーションミックスによりライゲートし、実証済みのREPLI-g テクノロジーをベースにした増幅ミックスを用いて増幅します。

図参照

Procedure.

アプリケーション

QuantiTect Whole Transcriptome Kit を用いて合成したcDNAはQuantiFastやRotor-Gene Kitsを用いたリアルタイムPCR解析に使用でき、将来行なう解析のために長期保存することもできます。このcDNAはマイクロアレイ解析には適切ではありません。少量あるいは貴重なサンプルからの全ゲノム増幅に、QIAGENはREPLI-g kitsを提供しています。配列によるバイアスは最小限で均一に増幅されるので、増幅したDNAはジェノタイピングやCGH（Comparative Genome Hybridization）のようなアプリケーションに最適です。

裏付けデータと数値

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
ApplicationsJA	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

リソース

For preparation of cDNA from total RNA by whole transcriptome amplification

Download Safety Data Sheets for QIAGEN product components.

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