Aerts Joeri L, Gonzales Monica I, Topalian Suzanne L
National Institutes of Health, Bethesda, MD, USA.
Biotechniques. 2004 Jan;36(1):84-6, 88, 90-1. doi: 10.2144/04361ST04.
Real-time reverse transcription PCR (RT-PCR) is a sensitive and accurate method to monitor gene expression and is often used to profile the expression of putative tumor antigens in the context of immunotherapy. However, this technique consists of several steps, including cell processing, RNA extraction, RNA storage, assessment of RNA concentration, and cDNA synthesis prior to PCR. To compensate for potential variability introduced in this procedure, the expression of housekeeping genes is commonly assessed in parallel with the expression of the gene of interest. In this study, the expression of a variety of housekeeping genes in a panel of 26 different human tumor and embryonal cell lines was assessed using real-time RT-PCR. For some control genes, the variability in expression was significant between different cell lines, despite the equalization of quantities of input RNA. The greatest variability was found for GAPDH. The lowest variability was found for beta-glucuronidase (GUS) and 18S rRNA. While real-time RT-PCR is a powerful tool for gene expression analysis, these results suggest that the choice of control genes to normalize the expression of the gene of interest is critical to the interpretation of experimental results and should be tailored to the nature of the study.
实时逆转录聚合酶链反应（RT-PCR）是一种监测基因表达的灵敏且准确的方法，常用于在免疫治疗背景下分析假定肿瘤抗原的表达情况。然而，该技术包含多个步骤，包括细胞处理、RNA提取、RNA储存、RNA浓度评估以及PCR前的cDNA合成。为补偿该过程中引入的潜在变异性，通常会同时评估管家基因的表达与感兴趣基因的表达。在本研究中，使用实时RT-PCR评估了26种不同人类肿瘤和胚胎细胞系中多种管家基因的表达。对于一些对照基因，尽管输入RNA的量已标准化，但不同细胞系之间的表达变异性仍然显著。GAPDH的变异性最大。β-葡萄糖醛酸酶（GUS）和18S rRNA的变异性最小。虽然实时RT-PCR是基因表达分析的有力工具，但这些结果表明，选择用于标准化感兴趣基因表达的对照基因对于实验结果的解释至关重要，应根据研究性质进行调整。
