Department of Microbiology, 32 Pearson Hall, Miami University, Oxford, OH 45056, USA.
J Virol Methods. 2010 Feb;163(2):210-5. doi: 10.1016/j.jviromet.2009.09.019. Epub 2009 Sep 23.
Quantitative real-time reverse transcription-PCR (qRT-PCR) has been used widely to measure gene transcription regulation in cells. qRT-PCR must include one or more internal housekeeping genes to normalize data collection. A strategy to use the host cell 28S rRNA as a housekeeping gene in qRT-PCR analysis of gene transcription of insect cells infected by baculovirus and ascovirus was developed. It has been found that the 28S rRNA reverse primer can be incorporated in the oligo-dT-primed cDNA synthesis reaction. In such a way, amplification of 28S cDNA showed lower and less variable cycle thresholds in cells infected by viruses than by using only oligo-dT and other published housekeeping genes such as the TATA box binding protein (TBP) gene, the peptidyl prolyl isomerase A (PPI) gene and the ribosomal protein 13 (L13) gene. Incorporation of the 28S reverse primer in oligo-dT-primed cDNA synthesis also does not interfere with the detection of other polymerase II transcribed genes.
实时荧光定量逆转录聚合酶链反应 (qRT-PCR) 已广泛用于测量细胞中的基因转录调控。qRT-PCR 必须包含一个或多个内参基因来对数据收集进行标准化。本研究开发了一种在杆状病毒和 ascovirus 感染的昆虫细胞基因转录的 qRT-PCR 分析中，将宿主细胞 28S rRNA 用作内参基因的策略。研究发现，28S rRNA 反转录引物可以整合到寡聚 dT- 引物 cDNA 合成反应中。通过这种方式，与仅使用寡聚 dT 和其他已发表的管家基因（如 TATA 框结合蛋白 (TBP) 基因、肽基脯氨酰异构酶 A (PPI) 基因和核糖体蛋白 13 (L13) 基因）相比，病毒感染细胞中 28S cDNA 的扩增显示出更低且更稳定的循环阈值。28S 反转录引物的整合也不会干扰其他聚合酶 II 转录基因的检测。
