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

  • 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

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。

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QuantiTect Rev. Transcription Kit (50)
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
205311
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QuantiTect Rev. Transcription Kit (200)
For 200 x 20 µl reactions: 4 x 100 µl 7x gDNA Wipeout Buffer, 4 x 50 µl Quantiscript Reverse Transcriptase, 4 x 200 µl 5x Quantiscript RT Buffer, 4 x 50 µl RT Primer Mix, 4 x 1.9 ml RNase-Free Water
205313
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QuantiTect Rev. Transcription Kit (400)
For 400 x 20 µl reactions: 800 µl 7x gDNA Wipeout Buffer, 400 µl Quantiscript Reverse Transcriptase, 1.6 ml 5x Quantiscript RT Buffer, 400 µl RT Primer Mix, 8 x 1.9 ml RNase-Free Water
205314
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The QuantiTect Reverse Transcription Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Fast and convenient cDNA synthesis.|Effective genomic DNA removal for accurate real-time RT-PCR.|Sensitive detection of a target at the 5' region of a 12.5 kb transcript.|Higher sensitivity in real-time, two-step RT-PCR.|
Genomic DNA removal and cDNA synthesis take only 20 minutes with the QuantiTect Reverse Transcription Kit. The procedure is fast and convenient since both reactions are run using the same incubation temperature and are set up using master mixes. In contrast, the procedure for the kit from Supplier I is much longer and requires more "hands-on time" due to additional pipetting steps and frequent changes in incubation temperature.|Real-time, two-step RT-PCR analysis of β-actin with (+RT) or without (-RT) reverse transcriptase. cDNA was synthesized from 100 ng total RNA, and real-time PCR was performed in duplicate on the LightCycler 2.0 using the QuantiTect SYBR® Green PCR Kit. The β-actin-specific primers detected both mRNA and genomic DNA sequences. Control reactions with no template were also performed (green). [A] The RT step was carried out using the QuantiTect Reverse Transcription Kit. The red, flat –RT plot indicates efficient removal of residual genomic DNA. [B] The RT step was carried out using a kit from Supplier I (Enzyme SIII). The purple –RT plot indicates amplification of residual genomic DNA.|Real-time, two-step RT-PCR of a target located at the 5' region of the mouse dystrophin gene (about 12.5 kb upstream of the poly-A site). Total RNA purified from mouse testis was reverse transcribed with the QuantiTect Reverse Transcription Kit as well as with reverse transcriptases from Supplier I and Supplier R. Identical volumes of triplicate reverse-transcription reactions were analyzed by real-time PCR on the LightCycler system. The error bars show the standard deviation for each set of triplicates. Compared with the other two kits, the QuantiTect Reverse Transcription Kit generated much higher amounts of cDNA (indicated by the lower CT values) and provided greater reproducibility in real-time RT-PCR (indicated by the smaller error bars). RFU: relative fluorescence units.

(Data kindly provided by Dr. Andrej-Nikolai Spiess, Department of Molecular Andrology, University Hospital Hamburg, Germany).|Real-time, two-step RT-PCR analysis of [A] TGFB2 (low expression) and [B] IL8 (higher expression). Total RNA was purified from human whole blood using the PAXgene Blood RNA system. cDNA was then synthesized from 1 µg RNA using the QuantiTect Reverse Transcription Kit, a kit from Supplier AII, or a kit from Supplier I. Real-time PCR was performed in duplicate on the ABI PRISM 7900 using the QuantiTect Probe PCR Kit and QuantiTect Gene Expression Assays for TGFB2 or IL8. The CT values for TGFB2 were lowest with the QuantiTect Reverse Transcription Kit, demonstrating that even low-abundance genes can be efficiently reverse transcribed and sensitively detected in real-time PCR.|

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。

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。

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。

Feature
Specifications
Applications Quantification of (even low-abundance) transcripts
Enzyme activity Reverse transcription
Mastermix No
Reaction type Two-step, cDNA production, genomic DNA digestion
Real-time or endpoint Real time
Sample/target type RNA template
Single or multiplex Single

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Kit Handbooks
1
For cDNA synthesis with integrated removal of genomic DNA contamination For use in real-time two-step RT-PCR
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Fast and convenient cDNA synthesis.
Fast and convenient cDNA synthesis.

Genomic DNA removal and cDNA synthesis take only 20 minutes with the QuantiTect Reverse Transcription Kit. The procedure is fast and convenient since both reactions are run using the same incubation temperature and are set up using master mixes. In contrast, the procedure for the kit from Supplier I is much longer and requires more "hands-on time" due to additional pipetting steps and frequent changes in incubation temperature.

Effective genomic DNA removal for accurate real-time RT-PCR.
Effective genomic DNA removal for accurate real-time RT-PCR.
Real-time, two-step RT-PCR analysis of β-actin with (+RT) or without (-RT) reverse transcriptase. cDNA was synthesized from 100 ng total RNA, and real-time PCR was performed in duplicate on the LightCycler 2.0 using the QuantiTect SYBR® Green PCR Kit. The β-actin-specific primers detected both mRNA and genomic DNA sequences. Control reactions with no template were also performed (green). [A] The RT step was carried out using the QuantiTect Reverse Transcription Kit. The red, flat –RT plot indicates efficient removal of residual genomic DNA. [B] The RT step was carried out using a kit from Supplier I (Enzyme SIII). The purple –RT plot indicates amplification of residual genomic DNA.
Senstitive detection of a target at the 5' region of a 12.5 kb transcript.
Sensitive detection of a target at the 5' region of a 12.5 kb transcript.

Real-time, two-step RT-PCR of a target located at the 5' region of the mouse dystrophin gene (about 12.5 kb upstream of the poly-A site). Total RNA purified from mouse testis was reverse transcribed with the QuantiTect Reverse Transcription Kit as well as with reverse transcriptases from Supplier I and Supplier R. Identical volumes of triplicate reverse-transcription reactions were analyzed by real-time PCR on the LightCycler system. The error bars show the standard deviation for each set of triplicates. Compared with the other two kits, the QuantiTect Reverse Transcription Kit generated much higher amounts of cDNA (indicated by the lower CT values) and provided greater reproducibility in real-time RT-PCR (indicated by the smaller error bars). RFU: relative fluorescence units.

(Data kindly provided by Dr. Andrej-Nikolai Spiess, Department of Molecular Andrology, University Hospital Hamburg, Germany).

Higher sensitivity in real-time two-step RT-PCR.
Higher sensitivity in real-time, two-step RT-PCR.
Real-time, two-step RT-PCR analysis of [A] TGFB2 (low expression) and [B] IL8 (higher expression). Total RNA was purified from human whole blood using the PAXgene Blood RNA system. cDNA was then synthesized from 1 µg RNA using the QuantiTect Reverse Transcription Kit, a kit from Supplier AII, or a kit from Supplier I. Real-time PCR was performed in duplicate on the ABI PRISM 7900 using the QuantiTect Probe PCR Kit and QuantiTect Gene Expression Assays for TGFB2 or IL8. The CT values for TGFB2 were lowest with the QuantiTect Reverse Transcription Kit, demonstrating that even low-abundance genes can be efficiently reverse transcribed and sensitively detected in real-time PCR.