Zhang Yi, Wei Zhenguo, Li Yuan-Yuan, Chen Yuhua, Shen Weide, Lu Changde
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, People's Republic of China.
Anal Biochem. 2009 Nov 15;394(2):202-8. doi: 10.1016/j.ab.2009.07.043. Epub 2009 Jul 30.
To quantify the transcription level of a gene, we have conceived a novel concept, transcription level of messenger RNA (mRNA) per gene copy, which was determined with a dual-spike-in strategy. In this strategy, an exogenous DNA was added as the spike reference for target DNA in addition to the exogenous RNA as the reference for target RNA. After the mRNA-to-DNA ratio of a target gene was estimated by real-time polymerase chain reaction (PCR), it was first normalized with the mRNA-to-DNA ratio of the exogenous reference. The normalized ratio was multiplied by the ratio of exogenous RNA to exogenous DNA to obtain the transcription level of mRNA per gene copy. This quantified transcription value allows one to compare the expression of a target gene in different tissues or the expression in a specified tissue under different conditions.
为了量化基因的转录水平，我们构思了一个新概念，即每个基因拷贝的信使核糖核酸（mRNA）转录水平，它是通过双内参策略确定的。在该策略中，除了添加外源RNA作为靶RNA的参照外，还添加了外源DNA作为靶DNA的加标参照。通过实时聚合酶链反应（PCR）估算出靶基因的mRNA与DNA的比率后，首先用外源参照的mRNA与DNA的比率进行标准化。将标准化后的比率乘以外源RNA与外源DNA的比率，以获得每个基因拷贝的mRNA转录水平。这个量化的转录值使人们能够比较靶基因在不同组织中的表达情况，或在不同条件下特定组织中的表达情况。
