QuantiTect Whole Transcriptome Kit
For unlimited real-time PCR analysis from precious RNA samples
- High cDNA yields for unlimited qPCR analysis and archiving
- Equal amplification of all cDNA and all transcript regions
- Fast and easy protocol with optimized reagents
The QuantiTect Whole Transcriptome Kit enables the preamplification and reverse transcription of limited amounts of RNA to high yields of cDNA for unlimited gene expression analysis using real-time PCR. An optimized protocol ensures a fast and easy procedure. The kit consists of a complete set of enzymes and buffers for whole transcriptome amplification (WTA). Innovative modification of Phi 29 polymerase technology allows yields of up to 40 μg cDNA from as little as 1 ng RNA. The uniquely high processivity of this polymerase guarantees the generation of cDNA containing uniformly amplified targets to ensure reliable gene expression analysis in real-time PCR.
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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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QuantiTect Whole Transcriptome Kit适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。
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
The QuantiTect Whole Transcriptome Kit provides highly uniform amplification of all transcripts, which is essential for reliable gene expression analysis. All mRNA transcripts are amplified with equal representation at both 5' and 3' regions (see figure "Equal amplification of 5' and 3' regions"). Preservation of the transcript profile is demonstrated in the figure "Preservation of transcript profile", where WTA-amplified cDNA prepared using the QuantiTect Whole Transcriptome Kit is compared with nonamplified cDNA prepared using a reverse transcriptase.
High and reproducible cDNA yields of up to 40 μg can be achieved (see figure "Reproducible cDNA yields"). This allows unlimited real-time PCR analysis with highly reproducible CT values (see table and figure "Reliable real-time PCR analysis"). In addition, since cDNA is more stable than RNA, it can be safely archived for future studies.
| 10 ng RNA |
2 hours |
Up to 10 µg cDNA |
1000 |
| 10 ng RNA |
8 hours |
Up to 40 µg cDNA |
4000 |
Principle
Gene expression profiling can be limited by the small amount of biological sample available. By using the QuantiTect Whole Transcriptome Kit, unlimited real-time PCR analyses of small and precious samples are possible. The QuantiTect Whole Transcriptome Kit is optimized for whole transcriptome amplification from as little as 1 ng RNA, or RNA corresponding to about 50 cells. Even lower amounts of RNA can be used, depending on the quality of the RNA and the abundance of the transcript of interest.
The QuantiTect Whole Transcriptome Kit contains reverse transcriptase, DNA polymerase, and optimized buffers and reagents for the amplification of all transcripts within an RNA sample. The kit integrates cDNA synthesis with the proven quality of REPLI-g technology for nonbiased sequence amplification. REPLI-g amplification uses Multiple Displacement Amplification (MDA) technology, which carries out isothermal sequence amplification using a uniquely processive DNA polymerase (see figure "Whole transcriptome amplification"). This technology ensures unbiased amplification of all transcript regions, even of 5' ends, providing unlimited cDNA highly suited for real-time PCR.
Procedure
Unlimited cDNA representing the entire transcriptome is prepared from total RNA in a simple 3-step protocol (see flowchart " Procedure"). RNA is first reverse transcribed into cDNA using an optimized reverse transcription mix containing T-Script Enzyme with random and oligo-dT primers. The synthesized cDNA is ligated using a high-efficiency ligation mix, and then amplified using an amplification mix based on proven REPLI-g technology.
Applications
cDNA prepared using the QuantiTect Whole Transcriptome Kit is intended for use in real-time PCR analysis with QuantiFast or Rotor-Gene Kits and can be archived for future analysis. The cDNA is not suitable for use in microarray analysis. For whole genome amplification from small or precious samples, QIAGEN offers REPLI-g kits and REPLI-g Service. Amplification is highly uniform with minimal sequence bias, providing DNA suitable for applications such as genotyping and Comparative Genome Hybridization (CGH).
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For preparation of cDNA from total RNA by whole transcriptome amplification
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图片
cDNA产量具有可重复性。
使用QuantiTect Whole Transcriptome Kit扩增不同血液样本的总RNA (10 ng) 2或8个小时。使用PicoGreen试剂测定cDNA产量。
对5'和3'端的均衡扩增。
使用QuantiTect Whole Transcriptome Kit扩增总RNA (1 ng)。然后使用10 ng cDNA、β-actin特异性引物和QuantiTect SYBR® Green PCR Kit进行real-time PCR。β-actin转录本的5'和3'区域对应的扩增片段具有相似的CT值,表明QuantiTect Whole Transcriptome Kit可针对所有转录本区域进行同等扩增。
实验流程。
采用3个连续反应:逆转录、连接和全转录组扩增,从纯化的RNA中扩增cDNA。
可靠的real-time PCR分析。
使用QuantiTect Whole Transcriptome Kit扩增重复的RNA样本(各10 ng)。然后在Mx3005P系统上使用10 ng cDNA和QuantiFast Probe PCR Kit对标示靶点进行real-time PCR。重叠的曲线表示高度可重复的全转录组扩增。ACTB:β-actin;HPRT1:次黄嘌呤磷酸核糖转移酶1。
全转录组扩增。
先利用模板RNA合成cDNA,然后进行连接(未显示)。REPLI-g DNA聚合酶沿cDNA模板链移动,替代互补链。被替代的链成为复制模板,生成高产量的cDNA。
保存转录本的特征。
使用QuantiTect Whole Transcriptome Kit扩增总RNA(1 ng)2小时(WTA 2 h)或8小时(WTA 8 h),然后使用10 ng cDNA对5个靶点进行real-time PCR。使用Sensiscript RT Kit逆转录RNA但不扩增(Non-WTA)作为对照,1/5的RT反应物用于real-time PCR。使用QuantiTect Probe PCR Kit进行real-time PCR。[A] 在WTA 2 h、WTA 8 h和Non-WTA样本中,5个转录本的相对基因表达水平相当(采用HPRT1标准化),如图中重叠的点所示。[B] WTA 8 h样本的ΔCT值(各靶点的CT值减去内对照 β-actin的CT值)(Y轴)与Non-WTA样本的ΔCT值(X轴)的曲线具有高线性度。[A] 和 [B] 中的数据证明QuantiTect Whole Transcriptome Kit可保护转录本的特征。TP53:肿瘤蛋白p53;NFKB1:B细胞κ轻肽基因增强子核因子1;HPRT1:次黄嘌呤磷酸核糖转移酶1;TBP:TATA盒结合蛋白;TNFRSF6B:肿瘤坏死因子受体超家族成员6b,诱骗受体。
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