Expression profiling of archival renal tumors by quantitative PCR to validate prognostic markets
Expression profiling of archival renal tumors by quantitative PCR to validate prognostic markets
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DOI：
10.2144/000112562
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发表时间：
2007-11-01
期刊：
影响因子：
2.7
通讯作者：
Kim, Hyung L.
中科院分区：
文献类型：
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作者：
Glenn, Sean T.;Jones, Craig A.;Kim, Hyung L.
福尔马林固定石蜡包埋（FFPE）组织通常由大多数病理部门保存，是发现临床有用生物标志物的广泛可用资源。我们描述了我们的方法，优化定量逆转录PCR（RT-PCR）的表达分析，使用冷冻和存档组织。评价常用参考基因在正常肾和肾透明细胞癌（RCC）中表达的稳定性。当使用FFPE RCC时，用于计算RT-PCR的标准化因子的最佳参考基因是ACTB、RPL 13 A、GUS、RPLP 0、HPRT 1和SDHA。当使用冷冻RCC时，最佳参考基因是ACTB、RPL 13 A和GUS，这证实了当使用来自冷冻肾肿瘤的完整RNA时，使用多个参考基因提高了准确性。在23个冷冻和FFPE肾肿瘤中测定了先前报道的在RCC中具有预后意义的16种标志物的表达，代表了一系列肿瘤分级和分期;冷冻和FFPE肿瘤中测量的表达的相关系数为0.921（P < 0.001）。当使用冷冻肿瘤时，所有标记物均预测存活，并且当FFPE肿瘤用作RNA来源时，16种标记物中的14种预测存活。优化的RT-PCR检测方法可以准确地测量大多数预后肿瘤标志物的表达。
Formalin-fixed paraffin-embedded (FFPE) tissues are routinely stored by most pathology departments and are a widely available resource for discovery of clinically useful biomarkers. We describe our method for optimizing quantitative reverse transcription PCR (RT-PCR) for expression analysis using frozen and archival tissue. Commonly used reference genes were evaluated for stability of expression in normal kidney and clear cell renal cell carcinoma (RCC). Optimal reference genes for calculating normalization factors for RT-PCR were ACTB, RPL13A, GUS, RPLP0, HPRT 1, and SDHA when using FFPE RCC. The optimal reference genes when using frozen RCC were ACTB, RPL13A,and GUS, confirming that use of multiple reference genes improves accuracy when intact RNA from frozen renal tumors are used. Expression of 16 markers previously reported to have prognostic significance in RCC was determined in 23 matching frozen and FFPE renal tumors, representing a range of tumor grades and stages; correlation coefficient for expression measured in frozen and FFPE tumors was 0.921 (P < 0.001). All markers predicted survival when frozen tumors were used and 14 of the 16 markers predicted survival when FFPE tumors were used as the source of RNA. An optimized RT-PCR assay can accurately measure expression of most prognostic tumor markers.