Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes
Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes
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DOI：
10.1038/labinvest.3700208
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发表时间：
2005-01-01
影响因子：
5
通讯作者：
Span, PN
中科院分区：
文献类型：
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作者：
de Kok, JB;Roelofs, RW;Span, PN
为了解释临床肿瘤样本中的定量基因表达测量结果，需要使用标准化器来校正样本之间细胞输入、RNA 质量和 RT 效率差异的表达数据。在许多研究中，使用单个管家基因进行标准化。然而，尚未鉴定出明确的单一参考基因（已证明细胞之间的表达不变）。作为最好的选择，多个管家基因的平均表达可用于标准化。在这项研究中，没有尝试确定标准化的金标准基因，而是确定可以准确替代多个基因测量的最佳单一管家基因。通过实时定量 RT-PCR，确定了来自结直肠、乳腺、前列腺、皮肤和膀胱组织的 80 个正常和肿瘤样本中 13 个常用管家基因的表达模式。这些基因包括大核糖体蛋白、β-肌动蛋白、亲环蛋白 A、3-磷酸甘油醛脱氢酶、磷酸甘油激酶 1、β-2-微球蛋白、β-葡萄糖醛酸酶、次黄嘌呤核糖基转移酶 (HPRT)、TATA 盒结合蛋白、转铁蛋白受体、胆色素原脱氨酶、ATP 合成酶 6 和 18S核糖体RNA。主成分分析用于分析这些表达模式，与表达水平无关。我们的方法将 HPRT 确定为单一最佳参考基因，可用作测量多个管家基因的准确且经济的替代方案。我们推荐该基因用于未来的研究，以标准化癌症研究和肿瘤诊断中的基因表达测量，直到确定明确的金标准。
For interpretation of quantitative gene expression measurements in clinical tumor samples, a normalizer is necessary to correct expression data for differences in cellular input, RNA quality, and RT efficiency between samples. In many studies, a single housekeeping gene is used for normalization. However, no unequivocal single reference gene ( with proven invariable expression between cells) has been identified yet. As the best alternative, the mean expression of multiple housekeeping genes can be used for normalization. In this study, no attempt was made to determine the gold-standard gene for normalization, but to identify the best single housekeeping gene that could accurately replace the measurement of multiple genes. Expression patterns of 13 frequently used housekeeping genes were determined in 80 normal and tumor samples from colorectal, breast, prostate, skin, and bladder tissues with real-time quantitative RT-PCR. These genes included, large ribosomal protein, beta-actin, cyclophilin A, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerokinase 1, beta-2-microglobin, beta-glucuronidase, hypoxanthine ribosyltransferase (HPRT), TATA-box-binding protein, transferrin receptor, porphobilinogen deaminase, ATP synthase 6, and 18S ribosomal RNA. Principal component analysis was used to analyze these expression patterns, independent of the level of expression. Our approach identified HPRT as the single best reference gene that could be used as an accurate and economic alternative for the measurement of multiple housekeeping genes. We recommend this gene for future studies to standardize gene expression measurements in cancer research and tumor diagnostics until a definite gold standard has been determined.