Selection of appropriate control genes to assess expression of tumor antigens using real-time RT-PCR
Selection of appropriate control genes to assess expression of tumor antigens using real-time RT-PCR
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DOI：
10.2144/04361st04
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发表时间：
2004-01-01
期刊：
影响因子：
2.7
通讯作者：
Topalian, SL
中科院分区：
文献类型：
--
作者：
Aerts, JL;Gonzales, MI;Topalian, SL
实时逆转录PCR（RT-PCR）是一种灵敏、准确的基因表达监测方法，常用于分析免疫治疗背景下推定肿瘤抗原的表达。然而，该技术由几个步骤组成，包括细胞处理、RNA提取、RNA储存、RNA浓度评估和PCR前的cDNA合成。为了补偿在该程序中引入的潜在可变性，通常与感兴趣的基因的表达平行地评估管家基因的表达。在这项研究中，使用实时RT-PCR评估了一组26种不同的人类肿瘤和胚胎细胞系中各种管家基因的表达。对于一些对照基因，尽管输入RNA的量相等，但不同细胞系之间表达的变异性是显著的。GAPDH的变异性最大。β-葡萄糖醛酸酶（GUS）和18 S rRNA的变异性最低。虽然实时RT-PCR是基因表达分析的强大工具，但这些结果表明，选择控制基因来标准化感兴趣基因的表达对于解释实验结果至关重要，并且应根据研究的性质进行定制。
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.