Siemens Healthcare Diagnostics Products, Molecular Research Germany, Cologne, Germany.
Clin Chem. 2010 Dec;56(12):1845-53. doi: 10.1373/clinchem.2010.151233. Epub 2010 Oct 14.
There is an increasing need for the identification of both DNA and RNA biomarkers from pathodiagnostic formalin-fixed paraffin-embedded (FFPE) tissue samples for the exploration of individualized therapy strategies in cancer. We investigated a fully automated, xylene-free nucleic acid extraction method for the simultaneous analysis of RNA and DNA biomarkers related to breast cancer.
We copurified both RNA and DNA from a single 10-μm section of 210 paired samples of FFPE tumor and adjacent normal tissues (1-25 years of archival time) using a fully automated extraction method. Half of the eluate was DNase I digested for mRNA expression analysis performed by using reverse-transcription quantitative PCR for the genes estrogen receptor 1 (ESR1), progesterone receptor (PGR), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (ERBB2), epoxide hydrolase 1 (EPHX1), baculoviral IAP repeat-containing 5 (BIRC5), matrix metallopeptidase 7 (MMP7), vascular endothelial growth factor A (VEGFA), and topoisomerase (DNA) II alpha 170kDa (TOP2A). The remaining undigested aliquot was used for the analysis of 7 single-nucleotide polymorphisms (SNPs) by MALDI-TOF mass spectrometry.
In 208 of 210 samples (99.0%) the protocol yielded robust quantification-cycle values for both RNA and DNA normalization. Expression of the 8 breast cancer genes was detected in 81%-100% of tumor tissues and 21%-100% of normal tissues. The 7 SNPs were successfully genotyped in 91%-97% of tumor and 94%-97% of normal tissues. Allele concordance between tumor and normal tissue was 98.9%-99.5%.
This fully automated process allowed an efficient simultaneous extraction of both RNA and DNA from a single FFPE section and subsequent dual analysis of selected genes. High gene expression and genotyping detection rates demonstrate the feasibility of molecular profiling from limited archival patient samples.
越来越需要从病理诊断的福尔马林固定石蜡包埋（FFPE）组织样本中鉴定 DNA 和 RNA 生物标志物，以探索癌症的个体化治疗策略。我们研究了一种全自动、无二甲苯的核酸提取方法，用于同时分析与乳腺癌相关的 RNA 和 DNA 生物标志物。
我们使用全自动提取方法从 210 对 FFPE 肿瘤和相邻正常组织（1-25 年存档时间）的 210 个 10μm 切片中共同纯化 RNA 和 DNA。一半洗脱液用 DNA 酶 I 消化，用于通过逆转录定量 PCR 分析基因雌激素受体 1 (ESR1)、孕激素受体 (PGR)、v-erb-b2 红细胞白血病病毒致癌基因同源物 2、神经/胶质母细胞瘤衍生致癌基因同源物（禽）(ERBB2)、环氧化物水解酶 1 (EPHX1)、杆状病毒 IAP 重复包含 5 (BIRC5)、基质金属蛋白酶 7 (MMP7)、血管内皮生长因子 A (VEGFA) 和拓扑异构酶（DNA）II alpha 170kDa (TOP2A) 的 mRNA 表达。剩余未消化的等分试样用于通过 MALDI-TOF 质谱分析 7 个单核苷酸多态性 (SNP)。
在 210 个样本中的 208 个样本（99.0%）中，该方案产生了 RNA 和 DNA 标准化的稳健定量周期值。8 个乳腺癌基因的表达在 81%-100%的肿瘤组织和 21%-100%的正常组织中均有检测到。7 个 SNP 在 91%-97%的肿瘤和 94%-97%的正常组织中成功进行了基因分型。肿瘤组织和正常组织之间的等位基因一致性为 98.9%-99.5%。
该全自动流程允许从单个 FFPE 切片中高效地共同提取 RNA 和 DNA，并随后对选定基因进行双重分析。高基因表达和基因分型检测率证明了从有限的存档患者样本中进行分子分析的可行性。
