Goidin D, Mamessier A, Staquet M J, Schmitt D, Berthier-Vergnes O
INSERM U 346, affiliée CNRS, Edouard Herriot Hospital, Lyon, F-69437, France.
Anal Biochem. 2001 Aug 1;295(1):17-21. doi: 10.1006/abio.2001.5171.
The comparison of the gene expression profiles between two subpopulations of melanoma cells (1C8 and T1C3) derived from the tumor of one patient by cDNA array revealed differences in GAPDH and beta-actin gene levels. These two housekeeping genes were up-regulated in invasive T1C3 melanoma cells compared to noninvasive 1C8 cells. Since cDNA array results were not confirmed by conventional RT-PCR throughout the exponential phase of amplification, we performed duplex relative RT-PCR using ribosomal 18S RNA as internal standard including competimer technology. Statistical analyses provided significant evidence that invasive T1C3 melanoma cells exhibited a twofold higher mRNA level of both GAPDH and beta-actin than noninvasive 1C8 cells. This study demonstrates that the duplex relative RT-PCR procedure including ribosomal 18S RNA as internal standard and competimer technology is precise for RNA quantification and is tailored for cDNA array validation. Our data provide molecular evidence that cellular subpopulations of the same pathological origin are highly heterogeneous and extend the concept that the selection of an appropriate internal control for comparative mRNA analysis should be adapted to each model of human cancers.
通过cDNA芯片对源自一名患者肿瘤的两个黑色素瘤细胞亚群（1C8和T1C3）的基因表达谱进行比较，结果显示甘油醛-3-磷酸脱氢酶（GAPDH）和β-肌动蛋白基因水平存在差异。与非侵袭性的1C8细胞相比，这两个管家基因在侵袭性T1C3黑色素瘤细胞中上调。由于在整个扩增指数期，传统逆转录-聚合酶链反应（RT-PCR）未证实cDNA芯片结果，因此我们使用核糖体18S RNA作为内参，包括竞争引物技术进行双重相对RT-PCR。统计分析提供了重要证据，表明侵袭性T1C3黑色素瘤细胞中GAPDH和β-肌动蛋白的mRNA水平均比非侵袭性1C8细胞高两倍。本研究表明，以核糖体18S RNA作为内参并采用竞争引物技术的双重相对RT-PCR程序在RNA定量方面是精确的，且适用于cDNA芯片验证。我们的数据提供了分子证据，表明相同病理来源的细胞亚群具有高度异质性，并扩展了这样一个概念，即选择合适的内参进行比较性mRNA分析应适用于每种人类癌症模型。
