从肺癌组织提取高纯度总RNA方法的优化及应用

Optimizing Method of High Quality Total RNA Isolated and Purified from Lung Cancer and Its Application

关键词:  肿瘤组织  非小细胞肺癌  RNA提取  TRIzol法  实时荧光定量PCR

Key words:  tumor tissue  non-small cell lung cancer  RNA extraction  TRIzol method  real-time quantitative PCR

摘要:

目的 通过对传统TRIzol提取组织中RNA的方法进行优化，建立一种较为简便、高效、经济的提取方法，并用优化后的方法检测非小细胞肺癌组织中ERCC1、RRM1和BRCA1的mRNA表达水平。方法 非小细胞肺癌的组织标本(包括肿瘤组织和正常组织)36例，采用传统和优化后的方法提取肺癌组织及正常组织中RNA，以SYBR Green荧光实时定量PCR分析各组织中ERCC1、RRM1、BRCA1以及管家基因GAPDH的mRNA表达水平。结果 传统方法的RNA浓度和纯度分别为(0.76±0.07)mg·mL^-1和1.87±0.04，优化方法的RNA浓度和纯度分别为(1.12±0.07)mg·mL^-1和1.95±0.03；琼脂糖凝胶电泳表明RNA没有降解，各个引物的熔解曲线均呈单峰，特异性良好。实时荧光定量显示，肺癌组织中ERCC1、RRM1和BRCA1的相对mRNA表达高于正常组织；ERCC1和BRCA1在不同病理类型(腺癌、鳞癌)的肿瘤组织中表达无显著性差异，RRM1在肺鳞癌中的表达显著高于腺癌(P<0.05)；不同性别中的基因表达均无显著性差异。结论 相比传统方法，利用优化后的方法提取样本的总RNA浓度、纯度和完整性均显著提高，能满足各种基因表达和定量检测实验的需要。非小细胞肺癌中ERCC1、RRM1和BRCA1的表达水平与肿瘤增殖活性密切相关，与临床肿瘤的病理类型也有一定的相关性。

Abstract:

OBJECTIVE To develop a more simple, efficient and economical method of total RNA extracted from human non-small cell lung cancer tissues by optimization of traditional TRIzol method, and detect mRNA expression levels of ERCC1, RRM1 and BRCA1. METHODS Thirty-six samples of non-small cell lung cancer(NSCLC, including tumor tissues and normal tissues) were collected and RNA from these samples were extracted through traditional and optimized method, expression levels of ERCC1, RRM1, BRCA1 and housekeeping gene GAPDH SYBR were dectected by RT-PCR. RESULTS The concentration and purity of RNA extracted by traditional method were (0.76±0.07)mg·mL^-1 and 1.87±0.04, respectively, the concentration and purity of RNA extracted by the optimized method performed better, which were (1.12±0.07)mg·mL^-1 and 1.95±0.03, respectively. The agarose gel electrophoresis showed no degradation of RNA, each primer melting curve showed a single peak and good specificity. According to the fluorescence-based real-time detection, ERCC1, RRM1 and BRCA1 mRNA levels in tumor tissues were respectively higher than those of normal tissues. No significance in expression of ERCC1 and BRCA1 between adenocarcinoma and squamous carcinoma was found. Nevertheless, RRM1 expression level in lung squamous carcinoma was significantly higher than that of adenocarcinoma(P<0.05). In addition, gene expression related to gender had no significant difference. CONCLUSION The concentration, purity and integrity of extracted total RNA meet the needs of a variety of gene expression and quantitative detection experiments. The expression levels of ERCC1, RRM1 and BRCA1 in NSCLC is closely correlated with tumor proliferative activity and clinical efficacy of adjuvant chemotherapy for cancer.