Measurement of gene expression in archival paraffin-embedded tissues - Development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay
Measurement of gene expression in archival paraffin-embedded tissues - Development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay
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DOI：
10.1016/s0002-9440(10)63093-3
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发表时间：
2004-01-01
影响因子：
6
通讯作者：
Baker, JB
中科院分区：
文献类型：
--
作者：
Cronin, M;Pho, M;Baker, JB
在整个名单的十年中，许多实验室已经表明，尽管在保存的组织中发生了广泛的RNA片段化，但福尔马林固定和石蜡包埋（FPE）组织标本中的mRNA水平可以通过逆转录酶-聚合酶链反应（RT-PCR）技术进行定量。我们已经开发了RT-PCR方法，灵敏，精确，并具有多分析物的能力，在临床研究和诊断分析的潜在广泛使用。这里显示，提取的FPE组织RNA的片段化程度随着存档存储时间显著增加。用于RT-PCR测定的探针和引物组基于短且长度均质的扩增子，能够实现有效的基于参考基因的数据标准化，用于年龄差异很大的标本的交叉比较。一个48个基因的检测用于比较来自相同的乳腺癌组织的基因表达谱，无论是冷冻或FPE显示非常相似的配置文件后，参考基因为基础的标准化。一个92个基因的检测，使用RNA提取的档案乳腺癌标本（从1985年至2001年）的三个10妈妈FPE部分产生了可分析的数据，这些基因在所有62个测试标本。结果基本一致时，雌激素受体，孕激素受体，和HER 2受体的状态确定的RT-PCR与这些受体的免疫组化检测进行了比较。此外，结果突出了RTPCR在定量和动态范围方面优于免疫组化。这些发现支持FPE组织RNA的RT-PCR分析作为多分析物临床诊断测试的平台的发展。
Throughout the List decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous hi length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10-mum FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RTPCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.