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
- 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
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。
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QuantiTect Whole Transcriptome Kit (25)
For 25 x 50 µl reactions: 25 µl T-Script Enzyme, 400 µl T-Script Buffer, 25 µl Ligation Enzyme 1, 25 µl Ligation Enzyme 2, 200 µl Ligation Reagent, 600 µl Ligation Buffer, 25 µl REPLI-g Midi DNA Polymerase, 730 µl REPLI-g Midi Reaction Buffer
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207043
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QuantiTect Whole Transcriptome Kit (100)
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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207045
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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.
Reproducible cDNA yields.|Equal amplification of 5' and 3' regions.|Procedure.|Reliable real-time PCR analysis.|Whole transcriptome amplification.|Preservation of transcript profile.|
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.|Total RNA (1 ng) was amplified using the QuantiTect Whole Transcriptome Kit. This was followed by real-time PCR using 10 ng cDNA, primers specific for β-actin, and the QuantiTect SYBR® Green PCR Kit. Amplicons corresponding to the 5' and 3' regions of the β-actin transcript were detected with similar CT values, indicating that the QuantiTect Whole Transcriptome Kit provided equal amplification of all transcript regions.|cDNA is amplified from purified RNA with 3 sequential reactions: reverse transcription, ligation, and whole transcriptome amplification.|Replicate RNA samples (10 ng each) were amplified using the QuantiTect Whole Transcriptome Kit. Real-time PCR of the indicated targets was then performed using 10 ng cDNA and the QuantiFast Probe PCR Kit on the Mx3005P system. The overlapping curves indicate highly reproducible whole transcriptome amplification. ACTB: β-actin; HPRT1: Hypoxanthine phosphoribosyltransferase 1.|cDNA is first synthesized from template RNA and then ligated (not shown). REPLI-g DNA polymerase moves along the cDNA template strand displacing the complementary strand. The displaced strand becomes a template for replication, allowing high yields of cDNA to be generated.|Total RNA (1 ng) was amplified using the QuantiTect Whole Transcriptome Kit for 2 hours (WTA 2 h) or 8 hours (WTA 8 h), followed by real-time PCR of 5 targets using 10 ng cDNA. As a control, RNA was reverse transcribed without amplification using the Sensiscript RT Kit (Non-WTA), and 1/5 of the RT reaction was used in real-time PCR. Real-time PCR was carried out using the QuantiTect Probe PCR Kit. [A] Relative gene expression levels of 5 transcripts (normalization to HPRT1) are similar in the WTA 2 h, WTA 8 h, and Non-WTA samples, as shown by the overlapping points. [B] A plot of ΔCT values (CT value for each target minus CT value for internal reference β-actin) for WTA 8 h samples (Y-axis) against those for Non-WTA samples (X-axis) is highly linear. The data in [A] and [B] demonstrate that the QuantiTect Whole Transcriptome Kit preserves the transcript profile. TP53: Tumor protein p53; NFKB1: nuclear factor of kappa light polypeptide gene enhancer in B-cells 1; HPRT1: hypoxanthine phosphoribosyltransferase 1; TBP: TATA box binding protein; TNFRSF6B: tumor necrosis factor receptor superfamily, member 6b, decoy.|
Performance
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、CT 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。
| 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 |
Principle
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。
Procedure
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 " 操作手順")。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。
Applications
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。
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For preparation of cDNA from total RNA by whole transcriptome amplification
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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.
Equal amplification of 5' and 3' regions.
Total RNA (1 ng) was amplified using the QuantiTect Whole Transcriptome Kit. This was followed by real-time PCR using 10 ng cDNA, primers specific for β-actin, and the QuantiTect SYBR® Green PCR Kit. Amplicons corresponding to the 5' and 3' regions of the β-actin transcript were detected with similar CT values, indicating that the QuantiTect Whole Transcriptome Kit provided equal amplification of all transcript regions.
Procedure.
cDNA is amplified from purified RNA with 3 sequential reactions: reverse transcription, ligation, and whole transcriptome amplification.
Reliable real-time PCR analysis.
Replicate RNA samples (10 ng each) were amplified using the QuantiTect Whole Transcriptome Kit. Real-time PCR of the indicated targets was then performed using 10 ng cDNA and the QuantiFast Probe PCR Kit on the Mx3005P system. The overlapping curves indicate highly reproducible whole transcriptome amplification. ACTB: β-actin; HPRT1: Hypoxanthine phosphoribosyltransferase 1.
Whole transcriptome amplification.
cDNA is first synthesized from template RNA and then ligated (not shown). REPLI-g DNA polymerase moves along the cDNA template strand displacing the complementary strand. The displaced strand becomes a template for replication, allowing high yields of cDNA to be generated.
Preservation of transcript profile.
Total RNA (1 ng) was amplified using the QuantiTect Whole Transcriptome Kit for 2 hours (WTA 2 h) or 8 hours (WTA 8 h), followed by real-time PCR of 5 targets using 10 ng cDNA. As a control, RNA was reverse transcribed without amplification using the Sensiscript RT Kit (Non-WTA), and 1/5 of the RT reaction was used in real-time PCR. Real-time PCR was carried out using the QuantiTect Probe PCR Kit. [A] Relative gene expression levels of 5 transcripts (normalization to HPRT1) are similar in the WTA 2 h, WTA 8 h, and Non-WTA samples, as shown by the overlapping points. [B] A plot of ΔCT values (CT value for each target minus CT value for internal reference β-actin) for WTA 8 h samples (Y-axis) against those for Non-WTA samples (X-axis) is highly linear. The data in [A] and [B] demonstrate that the QuantiTect Whole Transcriptome Kit preserves the transcript profile. TP53: Tumor protein p53; NFKB1: nuclear factor of kappa light polypeptide gene enhancer in B-cells 1; HPRT1: hypoxanthine phosphoribosyltransferase 1; TBP: TATA box binding protein; TNFRSF6B: tumor necrosis factor receptor superfamily, member 6b, decoy.
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