Xue Jian-Li, Cheng Xiao-Wen
Miami University, Oxford, Ohio, USA.
Curr Protoc Microbiol. 2010 Nov;Chapter 1:Unit1D.2. doi: 10.1002/9780471729259.mc01d02s19.
The use of quantitative real-time reverse transcription-PCR (qRT-PCR) for studying regulation of gene transcription requires an internal template-loading control or a housekeeping gene to guarantee the validity of the data collection, analysis, and interpretation. Analysis of gene transcription in virus-infected animal cells is problematic because virus infection often results in modified or fluctuating gene transcription patterns of conventionally used housekeeping genes, such as the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene and the β-actin gene. It has been demonstrated that the host 28S ribosomal gene can be used as a housekeeping gene in qRT-PCR in virus-infected insect cells. The stability of the human 28S rRNA gene transcription during the infection of HeLa cells with adenovirus has been confirmed, and this method has been extended to the use of the human 28S rRNA gene as a housekeeping gene in adenovirus-infected HeLa cells. Step-by-step instructions are described for use of this control in analysis of gene transcription in both types of virus-infected animal cells.
使用定量实时逆转录聚合酶链反应（qRT-PCR）研究基因转录调控时，需要一个内部模板加载对照或一个管家基因，以确保数据收集、分析和解释的有效性。分析病毒感染的动物细胞中的基因转录存在问题，因为病毒感染通常会导致传统使用的管家基因（如甘油醛-3-磷酸脱氢酶（GAPDH）基因和β-肌动蛋白基因）的基因转录模式发生改变或波动。已经证明，宿主28S核糖体基因可作为病毒感染昆虫细胞qRT-PCR中的管家基因。腺病毒感染HeLa细胞期间人28S rRNA基因转录的稳定性已得到证实，并且该方法已扩展到将人28S rRNA基因用作腺病毒感染的HeLa细胞中的管家基因。本文描述了在这两种病毒感染的动物细胞的基因转录分析中使用该对照的逐步说明。
