Sensitivity and reproducibility of standardized-competitive RT-PCR for transcript quantification and its comparison with real time RT-PCR.
Sensitivity and reproducibility of standardized-competitive RT-PCR for transcript quantification and its comparison with real time RT-PCR.
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DOI：
10.1186/1476-4598-3-5
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发表时间：
2004-01-23
期刊：
影响因子：
37.3
通讯作者：
Datar RH
中科院分区：
文献类型：
--
作者：
Pagliarulo V;George B;Beil SJ;Groshen S;Laird PW;Cai J;Willey J;Cote RJ;Datar RH
基于探针的检测测定形成转录物定量的支柱。这些测定的问题包括用于转录物定量的探针的不同杂交效率和所涉及的费用。我们检测了标准化竞争性RT-PCR（StaRT PCR）方法对两种细胞系（T24和LD 419）中4种细胞周期相关基因（RB、E2 F1、CDKN 2A和PCNA）转录本的定量能力，并与Taqman真实的时间定量RT-PCR方法进行了比较。我们还评估了StaRT PCR的灵敏度、重复性和一致性。StaRT PCR测定基于在PCR反应中沿着天然模板（NT）掺入精确标准化量的竞争性模板（CT）。这使得能够通过在PCR扩增结束时比较NT和CT条带强度来进行转录物定量。CT是理想的内部控制。转录本数量表示为对照基因（如β-肌动蛋白（ACTB））每百万个转录本的拷贝数。在整个StaRT PCR扩增循环中，NT和CT以非常相似的速率扩增，并且当NT/CT比率尽可能接近1：1时，变异系数最小（<3.8%）。在进行相同StaRT PCR反应的不同管中扩增速率之间的可变性非常低，并且在实验噪声范围内。此外，StaRT PCR足够灵敏以检测低至10%的内源性肌动蛋白转录物量的变化（通过配对学生t检验，p < 0.01）。StaRT PCR与Taqman真实的时间RT-PCR测定在转录物定量功效（通过斯皮尔曼秩相关方法，对于所有4个基因p < 0.01）和区分细胞类型和汇合模式的能力方面良好相关。因此，StaRT PCR是一种可靠和灵敏的技术，可应用于中高通量定量转录本测量。此外，在定量和区分能力方面，它与Taqman真实的时间PCR良好相关。这种无标记、廉价的技术可以提供产生个体肿瘤特有的重要分子特征的能力，并且可以鉴定新的治疗靶点。
Probe based detection assays form the mainstay of transcript quantification. Problems with these assays include varying hybridization efficiencies of the probes used for transcript quantification and the expense involved. We examined the ability of a standardized competitive RT-PCR (StaRT PCR) assay to quantify transcripts of 4 cell cycle associated genes (RB, E2F1, CDKN2A and PCNA) in two cell lines (T24 & LD419) and compared its efficacy with the established Taqman real time quantitative RT-PCR assay. We also assessed the sensitivity, reproducibility and consistency of StaRT PCR. StaRT PCR assay is based on the incorporation of competitive templates (CT) in precisely standardized quantities along with the native template (NT) in a PCR reaction. This enables transcript quantification by comparing the NT and CT band intensities at the end of the PCR amplification. The CT serves as an ideal internal control. The transcript numbers are expressed as copies per million transcripts of a control gene such as β-actin (ACTB). The NT and CT were amplified at remarkably similar rates throughout the StaRT PCR amplification cycles, and the coefficient of variation was least (<3.8%) when the NT/CT ratio was kept as close to 1:1 as possible. The variability between the rates of amplification in different tubes subjected to the same StaRT PCR reaction was very low and within the range of experimental noise. Further, StaRT PCR was sensitive enough to detect variations as low as 10% in endogenous actin transcript quantity (p < 0.01 by the paired student's t-test). StaRT PCR correlated well with Taqman real time RT-PCR assay in terms of transcript quantification efficacy (p < 0.01 for all 4 genes by the Spearman Rank correlation method) and the ability to discriminate between cell types and confluence patterns. StaRT PCR is thus a reliable and sensitive technique that can be applied to medium-high throughput quantitative transcript measurement. Further, it correlates well with Taqman real time PCR in terms of quantitative and discriminatory ability. This label-free, inexpensive technique may provide the ability to generate prognostically important molecular signatures unique to individual tumors and may enable identification of novel therapeutic targets.
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DOI：
10.1016/s0006-291x(02)00243-7
发表时间：
2002-04-26
影响因子：
3.1
作者：
Crawford, EL;Warner, KA;Willey, JC
通讯作者：
Willey, JC
影响因子：
2.7
作者：
Pfaffl, MW;Hageleit, M
通讯作者：
Hageleit, M
DOI：
10.1165/ajrcmb.19.1.3076
发表时间：
1998-07-01
影响因子：
6.4
作者：
Willey, JC;Crawford, EL;DeMuth, JP
通讯作者：
DeMuth, JP
DOI：
10.1054/modi.2001.29789
发表时间：
2001-12-01
期刊：
MOLECULAR DIAGNOSIS
影响因子：
--
作者：
Crawford, EL;Peters, GJ;Willey, JC
通讯作者：
Willey, JC
影响因子：
14.9
作者：
Yue, Huibin;Eastman, P. Scott;Johnston, Rick
通讯作者：
Johnston, Rick