一种新颖简便的荧光实时RT-PCR相对定量方法的建立

A Novel and Convenient Relative Quantitative Method of Fluorescence Real Time RT-PCR Assay Based on Slope of Standard Curve 稿件编号：2005-0257 中文关键词：荧光实时定量 RT-PCR ，相对定量，绝对定量，标准曲线， mRNA 定量，斜率 英文关键词：fluorescence real-time quantitative RT-PCR, relative quantification, absolute quantification, standard curve, mRNA quantification, slope 基金项目：江苏省高校自然科学指导计划资助项目（04KJD180044）和江苏大学高级人才科研启动基金资助项目（2281270002）. 作者单位 张驰宇 江苏大学医学技术学院，镇江 212001 徐顺高 江苏大学医学技术学院，镇江 212001 黄新祥 江苏大学医学技术学院，镇江 212001 中文摘要： 为建立一种新颖、简便的荧光实时 RT-PCR 相对定量方法，根据实时定量标准曲线，推导出相对定量基因表达的公式 . 公式显示相对表达指数只与 CT 值和标准曲线的斜率相关 . 构建标准曲线的标准品需要通过克隆和体外转录获得，实验过程繁琐 . 当人为成比例增减标准品各个稀释度的具体拷贝数时，标准曲线的斜率并不改变，说明标准曲线斜率与标准品的具体拷贝数无关 . 因此，新的相对定量方法可以用任何一个待测样品的总 RNA ( 或 cDNA) ，经系列稀释后作为标准品，来构建相对定量标准曲线，获得斜率 . 与绝对定量法比较，新方法获得了基本相同的斜率和非常一致的定量结果 ( 差异小于4%) ，而传统的 2 -ΔΔCT法却表现出较大的定量误差 . 这些结果表明，新的相对定量方法是一种简便、准确和高效的定量基因表达的方法 . 英文摘要： In order to develop a novel, simple and effective relative quantitative method using fluorescence real-time RT-PCR, the formulas were derived from the standard curve for quantifying relative expression of mRNA. The formulas show that the relative expression index is only associated with CT value and the slope of standard curve. RNA (DNA) standard for absolute quantitative standard curve is obtained generally by cloning and transcription in vitro. When input copy numbers of serial diluted RNA standards were increased or decreased n-fold (n > 0) proportionally, the slope of standard curve remains invariable, suggesting that the slope is independent of actual copy numbers of RNA standard. Therefore, anyone of tested RNA (cDNA) samples could be used after serial dilution as RNA standards to obtain slope. Compared with absolute quantitative method, the present one appears to have a more excellent consistency (difference less than 4%) in the relative expression indices with absolute quantitative method, than traditional relative method 2-ΔΔCT (difference more than 5%). The results show that the method described here is simple, precise and cost-effective for relative quantifying gene expression.