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分子生物学
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细胞生物学
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蛋白质组学
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蛋白凝胶纯化
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生化试剂
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仪器设备
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通用实验耗材
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色谱耗材
特点
- · 高cDNA产量用于无限次分析和长期保存
- · 均一扩增所有cDNA和所有转录本区段
- · 经优化的试剂，快速简单的操作流程
产品详情
QuantiTect Whole Transcriptome Kit对有限的RNA样品进行预扩增和逆转录，获得高产量cDNA，用于无限次real-time PCR基因表达分析。该试剂盒包含全转录扩增所需的全部酶和缓冲液。对Phi29聚合酶创新的修饰确保从低至1 ng RNA中制备多至40 μg cDNA。该聚合酶独特的加工过程可制备高度统一的cDNA，确保通过real-time PCR进行可靠的基因表达分析。
性能
QuantiTect Whole Transcriptome Kit确保所有转录子高度一致的扩增，这对于基因表达分析是必需的。所有mRNA转录子以相同的形式同时在5'和3'末端扩增（参见"Equal amplification of 5' and 3' regions.
Preservation of transcript profile.
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.
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.
可获得多至40 μg的可重复、高产量的cDNA（参见"Reproducible cDNA yields.
Reliable real-time PCR analysis.
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.
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.
| 起始材料 | 扩增时间 | 扩增产物的常规产量 | 可进行的real-time PCR次数* |
|---|---|---|---|
| 10 ng RNA | 2小时 | 至多10 µg cDNA | 1000 |
| 10 ng RNA | 8小时 | 至多40 µg cDNA | 4000 |
* 从一次QuantiTect whole transcriptome amplification (WTA)扩增反应得到的cDNA可进行的real-time PCR次数。不使用WTA，10 ng RNA只能进行一次可靠的real-time RT-PCR分析。
原理
获得的生物样本量太少可能限制基因表达谱分析。使用QuantiTect Whole Transcriptome Kit可对少量珍贵的样品进行无限次real-time PCR分析。QuantiTect Whole Transcriptome Kit经优化，用于1 ng RNA或相当于约50个细胞RNA的全转录扩增，甚至可以用更少量的RNA，这些取决于RNA的品质和转录子的丰度。
QuantiTect Whole Transcriptome Kit含有逆转录酶、DNA聚合酶和优化的缓冲液及试剂，可对一个RNA样本的所有转录本进行扩增。此试剂盒整合了经质量验证的REPLI-g技术，通过非偏向序列扩增来合成cDNA。REPLI-g扩增采用Multiple Displacement Amplification（MDA）技术，应用独特加工的DNA聚合酶实现等温扩增（参见下图）。该技术确保包括5’末端在内的所有转录区的同等扩增，从而获得可用于real-time PCR的足够多的cDNA。
操作流程
从总RNA开始经过3个步骤可获得大量代表了整个转录子的cDNA（参见" Procedure"）。首先，应用优化的含有T-Script Enzyme的逆转录混合物（含有随机引物和oligo-dT引物）将RNA转录为cDNA。用高效的连接混合物连接，然后用基于经验证的REPLI-g技术的扩增混合物扩增cDNA。
Procedure.
cDNA is amplified from purified RNA with 3 sequential reactions: reverse transcription, ligation, and whole transcriptome amplification.
应用
QuantiTect Whole Transcriptome Kit制备的cDNA可用于QuantiFast或Rotor-Gene Kits进行的real-time PCR分析，且可以储存用于以后的分析，但不适用于微阵列分析。QIAGEN提供REPLI-g Kits and Service，用于小量或珍贵样品的全基因组扩增。扩增高度一致，具有最小的序列偏好，可制备适合于基因分型和Comparative Genome Hybridization等应用的DNA。
参数
| Features | Specifications |
| Amplification | Whole total RNA |
| Starting material | Purified total RNA |
| Reaction time | 5 hours or 11 hours |
| Applications | 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 |
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