Application of transcript profiling in formalin-fixed paraffin-embedded diagnostic prostate cancer needle biopsies
Application of transcript profiling in formalin-fixed paraffin-embedded diagnostic prostate cancer needle biopsies
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DOI：
10.1111/j.1464-410x.2008.07627.x
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发表时间：
2008-08-01
影响因子：
4.5
通讯作者：
Gnanapragasam, Vincent J.
中科院分区：
文献类型：
--
作者：
Rogerson, Lynsey;Darby, Steve;Gnanapragasam, Vincent J.
为探讨转录本谱在前列腺癌福尔马林固定石蜡包埋（FFPE）诊断活检中的可行性，采用激光捕获显微切割（LCM）技术对前列腺穿刺活检标本中的腺上皮和间质组织进行显微切割。优化的RNA提取，逆转录和实时PCR（QPCR）方案用于检测转录本表达。使用水解的细胞系RNA和匹配的异种移植物FFPE和冷冻tumors.LCM和RNA提取在来自五名患者的试点队列的所有活检中实现的RNA降解效果进行了评估。所产生的cDNA成功地用于检测甘油醛-3-磷酸脱氢酶、RPL 13、前列腺特异性抗原、波形蛋白、分化抑制剂/DNA结合1（Id-1）和polycomb组蛋白增强子zeste同源物2（EZH 2）转录物的表达。在细胞系和异种移植模型中，我们研究了RNA降解对通过QPCR进行的转录物定量的影响。在这两种模型中，用管家基因对转录本数量进行标准化导致所有降解样品中的表达恢复至对照样品的50%差异内。使用29个活检样本的扩展队列，我们测试了在检测恶性和良性上皮之间EZH 2和Id-1表达差异中的应用。结果证实，我们的技术是能够量化的恶性和良性上皮细胞之间的表达显着差异与报告trends.This研究报告的使用标准FFPE针活检的转录谱和支持的概念，在前列腺癌诊断时获得的组织中的分子预后研究。
To investigate the feasibility of transcript profiling in diagnostic formalin-fixed and paraffin-embedded (FFPE) biopsies for prostate cancer.Laser-capture microdissection (LCM) was used to microdissect glandular epithelium as well as stromal tissue in archival prostate needle biopsies. Optimized RNA extraction, reverse transcription and real-time PCR (QPCR) protocols were used to detect transcript expression. RNA degradation effects were assessed using hydrolysed cell line RNA and matched xenograft FFPE and frozen tumours.LCM and RNA extraction was achieved in all biopsies from a pilot cohort of five patients. cDNA produced was successfully used to detect expression of glyceraldehyde-3-phosphate dehydrogenase, RPL13, prostate-specific antigen, vimentin, inhibitor of differentiation/DNA binding 1 (Id-1) and polycomb group protein enhancer of zeste homolog 2 (EZH2) transcripts. In the cell line and xenograft models, we investigated the effect of RNA degradation on transcript quantification by QPCR. In both models normalization of transcript quantity with a housekeeping gene resulted in restored expression in all degraded samples to within a 50% difference of control samples. Using an extended cohort of 29 biopsies, we tested application in detecting differences in EZH2 and Id-1 expression between malignant and benign epithelium. The results confirmed that our technique was capable of quantifying significant differences in expression between malignant and benign epithelium consistent with the reported trends.This study reports the use of standard FFPE needle biopsies for transcript profiling and supports the concept of molecular prognostic studies in tissue acquired at diagnosis in prostate cancer.