QuantiTect Reverse Transcription Kit

遺伝子発現解析のための高感度な2ステップリアルタイムRT-PCR用cDNAの迅速合成

S_1233_GEF_PCR0053
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QuantiTect Rev. Transcription Kit (50)

Cat. No. / ID:  205311

For 50 x 20 µl reactions: 100 µl 7x gDNA Wipeout Buffer, 50 µl Quantiscript Reverse Transcriptase, 200 µl 5x Quantiscript RT Buffer, 50 µl RT Primer Mix, 1.9 ml RNase-Free Water
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QuantiTect Reverse Transcription 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。
Need bulk, customized or optimized products for commercial purposes? We also offer support with logistics, compliance and more. Reach out to cooperate with QIAGEN Strategic Partnerships & OEM

Features

  • わずか20分でcDNA合成およびgDNA除去を完了
  • 低発現量の転写物からでも高収量でcDNAを合成
  • 転写物の5'および3'領域からでもcDNAを合成
  • RNA特異的なプライマーやプローブのデザインは不要

Product Details

QuantiTect Reverse Transcription KitはゲノムDNA除去を組み込んだcDNA合成を迅速かつ簡便な方法で実現します。RNAサンプル中のゲノムDNAコンタミはgDNA Wipeout Bufferで効率的に除去されます。Quantiscript Reverse Transcriptase、Quantiscript RT Buffer、画期的なRT Primer Mixにより、迅速かつ効率的な逆転写反応に必要な全ての成分が提供されます。合成されたcDNAはリアルタイムPCRでの使用に至適化され、mRNA転写物のどの領域の標的も確実に定量できます。

Performance

QuantiTect Reverse Transcription Kitを用いることにより、RNAサンプル中に混入しているゲノムDNAはユニークなgDNA Wipeout Bufferで効率的かつ迅速に除去されます(図" 効果的なゲノムDNA除去による正確なリアルタイムRT-PCR")。ゲノムDNAの排除は正確な遺伝子発現結果を得るために極めて重要であり、RNA特異的プライマーまたはプローブのデザインは必ずしも可能とは限りません。gDNA Wipeout Bufferを用いると、RNAサンプルの精製中あるいは精製後に別途にDNase分解を行なう必要がないため、時間および経費を節約できます。 

Quantiscript RT Bufferと組み合わせて用いることにより、Quantiscript Reverse Transcriptaseの高いRNAアフィニティーはどのようなRNAテンプレートからも高い収量でcDNAを合成できます(表"より少量の転写物からより高いcDNA収量")。GC含有率が高いあるいは複雑な二次構造を持つ逆転写が困難なテンプレートでさえも問題なく逆転写できます。

より少量の転写物からより高いcDNA収量
IL12AのCT
(低発現量)
IL1RNのCT
(高発現量)
スタートRNA(ng) QIAGEN Supplier AII QIAGEN Supplier AII
1000 30.9 32.0 23.1 24.9
100 34.2 35.4 26.3 26.6
10 37.8 46.8 29.7 30.3
1 非検出 非検出 32.4 34.5
種々のスタートRNA量を用いたIL12AとIL1RNの2ステップリアルタイムRT-PCR解析。トータルRNAからQuantiTect Reverse Transcription Kit あるいはSupplier AIIのキットを用いて逆転写反応を行なった。合成したcDNAをQuantiTect Probe PCR KitとIL12AあるいはIL1RN用QuantiTect Gene Expression Assayを用いてABI PRISM 7900上で解析した。QIAGENキットでのより低いCT値は(特に発現量が中等度のIL12A遺伝子、太字)、より高いcDNA収量を示唆している。N.D.:検出されなかった。

RT Primer Mixには全てのRNA転写物の5'領域も含むあらゆる領域からcDNA合成を実現する特別に至適化されたoligo-dTオリゴ-dTとランダムプライマーのミックスが含まれています(図" 12.5 kbの転写物の5'領域における標的を高感度で検出")。他社のキットと比較して、QuantiTect Reverse Transcription Kitは増幅する標的領域が転写物のどこに局在しているかに関係なく、リアルタイムPCR解析用のcDNAテンプレートを高い収量で合成し、発現量が少ない遺伝子の検出感度を上昇させます(図" より高い感度の2ステップリアルタイムRT-PCR")。また、QuantiTect Reverse Transcription Kitは、再現性のより高いリアルタイムRT-PCRを実現します。

See figures

Principle

QuantiTect Reverse TranscriptaseはOmniscriptとSensiscript Reverse Transcriptaseのブレンドで、RNAに対して高いアフィニティーを有し、幅広いRNA量(10 pg~1 µg)からcDNA合成が可能です。他社のキットと比較して、QuantiTect Reverse Transcription Kitは増幅する標的領域が転写物のどこに局在しているかに関係なく、リアルタイムPCR解析用のcDNAテンプレートを高い収量で合成します。GC含有率が高いあるいは複雑な二次構造を持つ逆転写が困難なテンプレートでさえも問題なく逆転写できます。また、QuantiTect RT BufferはリアルタイムPCRバッファーと互換性があるようにも至適化されています。

リアルタイムRT-PCRによる遺伝子発現アッセイで正確な結果を得るためには、cDNAのみを増幅し検出することが重要です。ゲノムDNAによるアッセイへの悪影響はエクソン/エクソン境界にまたがるプライマーやプローブをデザインすることにより避けることができます。しかし、この方法が不可能な場合もあり(例えばシングルエクソン遺伝子からのcDNA)、スタートとなるRNAサンプルがゲノムDNAを含まないことが必須です。QuantiTect Reverse Transcription Kitを用いることにより、RNAサンプル中に混入しているゲノムDNAはユニークなgDNA Wipeout Bufferで効率的かつ迅速に除去されます。RNAサンプルの精製中あるいは精製後に別途にDNase分解を行なう必要がないため、時間および経費を節約できます。RNA特異的なプライマーやプローブのデザインも不要です。 

QuantiTect Reverse Transcriptase Kitの構成
構成利点
gDNA Wipeout BufferリアルタイムRT-PCRのみでRNAを検出
Quantiscript Reverse Transcriptase幅広いRNA量(10 pg~1 µg RNA)の使用
高感度
Quantiscript RT Buffer増幅困難なテンプレートの読み取り
RT Primer Mix転写物の全ての領域、5'領域からもcDNAを合成

Procedure

QuantiTect Reverse Transcription Kitを用いれば、ゲノムDNA除去とcDNA合成はわずか20分で完了します(フローチャート" 迅速で簡便なcDNA合成")。両反応とも同一のインキュベーション温度で行なうことができ、マスターミックスによるセットアップができるため、操作は迅速かつ簡便です。

QuantiTect Reverse Transcription Kitには、迅速なcDNA合成に必要なものが全て含まれています。精製したRNAはgDNA Wipeout Bufferで短時間インキュベートし、混入したゲノムDNAを効率的に除去します。他の方法と比較し、逆転写反応において、Quantiscript Reverse Transcriptase、Quantiscript RT Buffer、RT Primer Mixで調製したマスターミックスを用い、RNAサンプルを直接使用します。Quantiscript Reverse Transcriptaseを用いると、複雑な2°構造でもRNAは低温で転写され、RNAは分解されません。反応全体は42℃で起こり、95℃で不活化されます。RNA変性、プライマーアニーリング、RNase H分解の追加ステップは必要ありません。

See figures

Applications

QuantiTect Reverse Transcription Kitはレーザーマイクロダイセクションサンプルや組織生検を含む全ての種類のスタートサンプルから感度および効率の良いリアルタイムRT-PCRを実現します。

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsQuantification of (even low-abundance) transcripts
Sample/target typeRNA template
Enzyme activityReverse transcription
Real-time or endpointReal time
Reaction typeTwo-step, cDNA production, genomic DNA digestion
Single or multiplexSingle
MastermixNo

Resources

テクニカルインフォメーション (1)
MSDS (1)
Download Safety Data Sheets for QIAGEN product components.
クイックスタートプロトコール (1)
パンフレット (1)
キットハンドブック (1)
For cDNA synthesis with integrated removal of genomic DNA contamination For use in real-time two-step RT-PCR
Certificates of Analysis (1)
Gene Expression Analysis (1)
Kit Handbooks (1)
For cDNA synthesis with integrated removal of genomic DNA contamination For use in real-time two-step RT-PCR
Quick-Start Protocols (1)

FAQ

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
Is it possible to use the QuantiTect Reverse Transcription Kit with bacterial RNA?

Yes, it is possible to use the QuantiTect Reverse Transcription Kit for bacteria. The RT Primer Mix provided in the kit is a unique, optimized blend of random primers and oligo-dT allowing high cDNA yields from all regions of RNA transcripts. It has successfully been tested for Reverse Transcription in bacteria as well. We strongly recommend to isolate bacterial RNA using the RNeasy Mini Kit prior to performing Reverse Transcription. This will ensure the high prep quality necessary for optimal RT results with the QuantiTect Reverse Transcription Kit.

FAQ ID -785
Is mRNA isolation necessary for sensitive RT-PCR?
Usually not. Since RT-PCR is extremely sensitive, as little as 10–200 ng total RNA is sufficient for each 25–50 µl reaction mix, depending on the RT system. For abundant mRNA species, it is possible to use even less than 10 ng total RNA. For rare mRNA species, use a sequence-specific primer in the RT reaction to increase sensitivity. RNA content in various cells and tissues can be found here.
FAQ ID -111
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 the Reverse Transcriptases of the QuantiTect Reverse Transcription Kit and the QuantiTect Probe RT-PCR Kit be used interchangeably?

No, please do not exchange Quantiscript Reverse Transcriptase of the QuantiTect Reverse Transcription Kit with QuantiTect RT Mix of the QuantiTect Probe RT-PCR Kit.

Although both are an optimized mixture of Omniscript and Sensiscript Reverse Transcriptases, the mixture provided in the QuantiTect Reverse Transription Kit is optimized for random priming in a two-step reaction, whereas the mixture in the QuantiTect Probe RT-PCR Kit is optimized for gene-specific priming in a one-step RT-PCR reaction.

 

FAQ ID -1066
Can I use my gene-specific primers with the FastLane Cell cDNA Kit and the QuantiTect Reverse Transcription Kit?

Yes, you can substitute the RT Primer Mix supplied in the FastLane Cell cDNA Kit and the QuantiTect Reverse Transcription Kit with your gene-specific primers. We suggest optimizinig the primer concentration by titration, starting at 1 uM, and gradually decreasing it to 0.5 uM final concentration in the reaction. Optimal amounts will depend on the specific primers you are using.

FAQ ID -812
What is the recommended solution in which to store RNA samples that will be used as templates for cDNA synthesis?
For best results, all RNA samples should be suspended in RNase-free water. Alternatively, RNase-free 1 mM sodium citrate (pH 6.5) or 10 mM Tris buffer (pH 7.0) may be used. Do not use DEPC-treated water, as most DEPC preparations are contaminated with molecules that are inhibitory to reverse transcription and/or PCR. For long-term storage, RNA preps may be stored at -70 ºC in RNase-free water, or the buffers listed above, or precipitated in ethanol or isopropanol. In order to avoid repeated freeze-thaw cycles, it is recommended that frozen RNA samples be stored as multiple, single-use aliquots.
FAQ ID -2659
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
How do the FastLane Cell cDNA Kit and QuantiTect Reverse Transcription Kit eliminate genomic DNA contamination?
The FastLane Cell cDNA Kit and QuantiTect Reverse Transcription Kit contain a unique buffer, called gDNA Wipeout Buffer, which ensures complete removal of gDNA after a brief incubation step.
FAQ ID -783
What is the threshold cycle or Ct value?
The Ct or threshold cycle value is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal significantly above the background fluorescence. At the threshold cycle, a detectable amount of amplicon product has been generated during the early exponential phase of the reaction. The threshold cycle is inversely proportional to the original relative expression level of the gene of interest.
FAQ ID -2682
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 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
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
Is it possible to scale up QuantiTect Reverse Transcription reactions to allow use of larger amounts of RNA?
Yes, reactions using the QuantiTect Reverse Transcription Kit can be scaled up. Please scale up all reaction components proportionally.
FAQ ID -1063
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
Do QuantiTect Primer Assays contain SYBR Green dye?

No, QuantiTect Primer Assays are supplied as lyophilized, premixed primer pairs. Reaction components for SYBR Green real-time RT-PCR must be purchased separately.

To find a QuantiTect Primer Assay for your target gene of interest, please visit our GeneGlobe data base.

FAQ ID -1143
Why should DNA or cDNA targets be less than 250 bp long for real-time PCR?

Shorter amplification products facilitate high PCR efficiencies. Ideally, amplicon length should be less than 150 bp for optimal amplification efficiency. PCR efficiencies close to 100% are a crucial prerequisite for accurate quantification of target copy numbers in real-time PCR.

FAQ ID-751
What are the recommended storage conditions for the QuantiTect Reverse Transcription Kit and its components?

The QuantiTect Reverse Transription Kit should be stored at -20°C immediately upon receipt. We recommend to aliquot the RT Primer Mix and keep it at -20°C.

If the kit is being used routinely, it may be convenient to prepare a premix of RT Primer Mix and 5x Quantiscript RT Buffer at a ratio of 1:4. Aliquot the premix and store at -20°C.

FAQ ID -1077
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