Hayward A L, Oefner P J, Sabatini S, Kainer D B, Hinojos C A, Doris P A
Institute of Molecular Medicine, University of Texas Health Science Center, 2121 West Holcombe Boulevard, Houston, TX 77030, USA.
Nucleic Acids Res. 1998 Jun 1;26(11):2511-8. doi: 10.1093/nar/26.11.2511.
The present studies demonstrate a theoretical and practical framework for the accurate quantitation of gene expression in RNA extracted from microscopic tissue samples. The approaches are developed around competitive RT-PCR techniques. Assay performance has been examined and validated at both the RT and PCR steps. Our analysis of RT transcription efficiency for a number of native and competitor combinations shows that this property can differ, even for very similar templates. However, this difference is consistent and, once identified and measured, can be removed as an obstacle to accuracy. Using mathematical modeling, we have examined the simulated co-amplification of native and competitor templates in PCR. Useful insights have emerged from such modeling which indicate that differences in initial amplification efficiency and the rate of decay of amplification efficiency during the reaction can rapidly lead to inaccuracy, even while the slope and linearity of log plots of the competitor input and reaction product ratios are close to ideal. Finally, we show here that competitive RT-PCR reactions do not have to remain in the log-linear phase of PCR in order to accomplish accurate and precise quantification. Using appropriate competitors sharing primer binding sites and high internal sequence similarity, identical amplification efficiencies are preserved throughout the reaction. Reaction products, including heteroduplexes formed between native and competitor templates as reactions progress to plateau, can be identified and quantified accurately using the new technique of denaturing HPLC (dHPLC). This analytical technique allows the accuracy of competitive RT-PCR to be preserved beyond the linear phase. The technique has high sensitivity and precision and target abundances as low as 100 copies could be reliably estimated.
目前的研究展示了一个理论和实践框架，用于从微观组织样本中提取的RNA中准确定量基因表达。这些方法是围绕竞争性逆转录聚合酶链反应（RT-PCR）技术开发的。在逆转录（RT）和聚合酶链反应（PCR）步骤均对分析性能进行了检测和验证。我们对多种天然模板和竞争模板组合的RT转录效率分析表明，即使对于非常相似的模板，这种特性也可能不同。然而，这种差异是一致的，一旦被识别和测量，就可以作为准确性的障碍被消除。通过数学建模，我们研究了PCR中天然模板和竞争模板的模拟共扩增。这种建模产生了有用的见解，表明初始扩增效率的差异以及反应过程中扩增效率的衰减速率会迅速导致不准确，即使竞争模板输入与反应产物比率的对数图的斜率和线性接近理想状态。最后，我们在此表明，竞争性RT-PCR反应不必停留在PCR的对数线性阶段就能实现准确和精确的定量。使用共享引物结合位点且内部序列相似度高的合适竞争模板，整个反应过程中能保持相同的扩增效率。随着反应进入平台期，反应产物，包括天然模板和竞争模板之间形成的异源双链体，可以使用变性高效液相色谱（dHPLC）新技术准确识别和定量。这种分析技术使竞争性RT-PCR的准确性在超出线性阶段后仍得以保持。该技术具有高灵敏度和高精度，低至100个拷贝的靶标丰度也能可靠估计。
