Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue
Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue
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DOI：
10.1016/s0002-9440(10)63985-5
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发表时间：
2001-02-01
影响因子：
6
通讯作者：
Höfler, H
中科院分区：
文献类型：
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作者：
Specht, K;Richter, T;Höfler, H
福尔马林固定、石蜡包埋的组织是回顾性临床研究中最广泛使用的材料。结合基因组学的潜力，这些组织代表了一个宝贵的资源，阐明疾病机制和验证差异表达的基因作为新的治疗靶点或预后指标。我们在这里描述了一种方法，结合激光辅助显微切割，允许在福尔马林固定，石蜡包埋的档案组织中进行定量基因表达分析。使用优化的RNA微量提取程序结合实时定量逆转录聚合酶链反应的荧光TaqMan方法，我们分析了一组癌症相关基因的表达，EGF-R，HER-2/neu，FGF-R4，p21/WAF 1/Cip 1，MDM 2，HPRT和PGK作为对照。我们证明，从福尔马林固定，石蜡包埋的组织的表达水平测定是准确的酸重现性，测量与匹配的新鲜冷冻组织获得的那些，固定等级和时间都没有显着影响的结果。用5 μ m厚的切片和确定数量的肿瘤细胞进行的激光显微切割研究表明，仅用50个细胞就可以实现特异性mRNA的可重复定量，我们将我们的方法应用于HER-2/在54例Barrett食管腺癌患者的显微切割肿瘤和非肿瘤存档样本中进行neu定量基因表达分析，结果显示与荧光平行获得的结果相匹配。原位杂交和免疫组织化学。因此，激光辅助显微切割和实时TaqMan逆转录酶-聚合酶链反应的结合为档案组织标本中基因表达变化的调查和临床验证开辟了新的途径。
Formalin-fixed, paraffin-embedded tissue is the most widely available material for retrospective clinical studies. In combination with the potential of genomics, these tissues represent an invaluable resource for the elucidation of disease mechanisms and validation of differentially expressed genes as novel therapeutic targets or prognostic indicators. We describe here an approach that, in combination with laser-assisted microdissection allows quantitative gene expression analysis in formalin-fixed, paraffin-embedded archival tissue. Using an optimized RNA microscale extraction procedure in conjunction with real-time quantitative reverse transcriptase-polymerase chain reaction based on fluorogenic TaqMan methodology, we analyzed the expression of a panel of cancer-relevant genes, EGF-R, HER-2/neu, FGF-R4, p21/ WAF1/Cip1, MDM2, and HPRT and PGK as controls. We demonstrate that expression level determinations from formalin-fixed, paraffin-embedded tissues are accurate acid reproducible, Measurements were comparable to those obtained with matching fresh-frozen tissue and neither fixation grade nor time significantly affected the results. Laser microdissection studies with 5-mum thick sections and defined numbers of tumor cells demonstrated that reproducible quantitation of specific mRNAs can be achieved with only 50 cells, We applied our approach to HER-2/neu quantitative gene expression analysis in 54 microdissected tumor and nonneoplastic archival samples from patients with Barrett's esophageal adenocarcinoma and showed that the results matched those obtained in parallel by fluorescence in situ hybridization and immunohistochemistry. Thus, the combination of laser-assisted microdissection and real-time TaqMan reverse transcriptase-polymerase chain reaction opens new avenues for the investigation and clinical validation of gene expression changes in archival tissue specimens.