Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR
Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR
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DOI：
10.1016/s1525-1578(10)60646-0
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发表时间：
2001-02-01
影响因子：
4.1
通讯作者：
Unger, ER
中科院分区：
文献类型：
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作者：
Rajeevan, MS;Vernon, SD;Unger, ER
我们评估了实时（动力学）逆转录聚合酶链式反应 (RT-PCR)，以验证 DNA 阵列识别的差异表达基因。使用人乳头瘤病毒物理状态（游离型或整合型）不同的两个角质形成细胞亚克隆的基因表达作为模型系统。高密度过滤阵列鉴定出 588 个基因中的 444 个为阴性或表达差异小于两倍，另外 144 个基因为独特表达或两个亚克隆之间差异超过两倍，实时 RT-PCR 使用基于 LightCycler 的 SYBR Green I 染料检测和熔解曲线分析来验证基因表达的相对变化。实时 RT-PCR 证实了高密度过滤阵列识别的 24 个基因中的 17 个（71%）基因的表达变化。具有强杂交信号和至少两倍差异的基因可能通过实时 RT-PCR 进行验证。该数据表明，(i) 杂交强度和差异表达水平决定了验证高密度过滤阵列结果的可能性，以及 (ii) 由 DNA 阵列识别出的具有两到四倍表达差异的基因不能在未经验证的情况下被消除为错误，也不能被接受为正确的。基于 Light-Cycler 技术的实时 RT-PCR 非常适合验证 DNA 阵列结果，因为它定量、快速，并且所需的 RNA 比传统检测少 1000 倍。
We evaluated real-time (kinetic) reverse transcription-polymerase chain reaction (RT-PCR) to validate differentially expressed genes identified by DNA arrays. Gene expression of two keratinocyte subclones differing in the physical state of human papillomavirus (episomal or integrated) was used as a model system. High-density filter arrays identified 444 of 588 genes as either negative or expressed with less than twofold difference, and the other 144 genes as expressed uniquely or with more than two-fold difference between the two subclones, Real-time RT-PCR used LightCycler-based SYBR Green I dye detection and melting curve analysis to validate the relative change in gene expression. Real-time RT-PCR confirmed the change in expression of 17 of 24 (71%) genes identified by high-density filter arrays. Genes with strong hybridization signals and at least twofold difference were likely to be validated by real-time RT-PCR This data suggests that (i) both hybridization intensity and the level of differential expression determine the Likelihood of validating high-density filter array results and (ii) genes identified by DNA arrays with a two- to fourfold difference in expression cannot be eliminated as false nor be accepted as true without validation. Real-time RT-PCR based on Light-Cycler technology is well-suited to validate DNA array results because it is quantitative, rapid, and requires 1000-foId less RNA than conventional assays.