胰腺癌及癌旁组织中提取RNA样品的方法优化及鉴定  

Optimization of the Purification Method of Total RNA from Pancreatic Cancer and Adjacent Tissues

作　　者：魏伟[1] 欧政林 窦晓淋 唐翎[2,3] WEIWei;OU Zheng-li;DOU Xiao-lin;TANG Ling(Department of General Surgery,Xiangya Hospital,Central South University,Changsha,Hunan,410008,China;Department of Pharmacy,Xiangya Hospital,Central South University,Changsha,Hunan,410008,China;National Clinical Research Center for Geriatric Disorders,Xiangya Hospital,Central South University,Changsha,Hunan,410008,China)

机构地区：[1]中南大学湘雅医院普外科,湖南长沙410008 [2]中南大学湘雅医院药学部,湖南长沙410008 [3]中南大学湘雅医院国家老年疾病临床医学研究中心,湖南长沙410008

出　　处：《现代生物医学进展》2022年第20期3813-3817,共5页Progress in Modern Biomedicine

基　　金：湖南省自然科学基金项目(2019JJ40489)。

摘　　要：目的:优化胰腺癌及癌旁组织总RNA的提取方法,为胰腺相关疾病的发病机制研究提供高质量的实验样本。方法:采用组织块分离剪碎、RNase清洗及抑制等方法对胰腺癌及癌旁组织进行预处理,液氮研磨及trizol-氯仿抽提的方法提取组织RNA。通过琼脂糖凝胶电泳及生物分析仪鉴定RNA的完整性。使用等量RNA作为逆转录模板,以oligo dT引物对信使RNA(message RNA,mRNA)进行逆转录;以茎环结构引物对微小RNA(microRNA,miRNA)进行逆转录。通过定量PCR的方法检测mRNA及miRNA的表达水平。结果:相较于常规方法,优化方法提取的RNA样品降解程度低,完整性较高。相对于胰腺癌组织,癌旁组织RNA更易降解,mRNA的表达水平出现降低趋势;但miRNA的表达在胰腺癌及癌旁组织中无明显差异。结论:胰腺癌及癌旁组织的预处理可降低RNA降低程度,可为RNA的表达检测提供高质量的实验样本,增加实验准确度。Objective:To optimize the extraction method of total RNA from pancreatic cancer and adjacent tissues,and provide high-quality experimental samples for the pathogenesis of pancreatic-related diseases.Methods:The pretreatment of pancreatic cancer and paracancerous tissues was accomplished through shredding,RNase-free washing and RNase inhibition.RNA was isolated by liquid nitrogen grinding and trizol-chloroform extraction.The integrity of RNA was identified by agarose gel electrophoresis and bioanalyzer.With the same amount of RNA as the reverse transcription template,oligo dT primers were used for reverse transcription of messenger RNA(mRNA);stem-loop structure primers were used to reverse transcription of microRNA(miRNA).The expression levels of mRNA and miRNA were detected by quantitative RT-PCR.Results:Compared with conventional methods,RNA samples extracted by the optimized method had a lower degradation degree and higher integrity.Compared with pancreatic cancer tissue,RNA in adjacent tissue is more easily degraded,and the expression level of mRNA tends to decrease.However,there is no significant difference in miRNA expression between pancreatic cancer and adjacent tissue.Conclusions:Pretreatment of pancreatic cancer and adjacent tissues could reduce the RNA degradation,and provide high-quality experimental samples for RNA investigation and high experimental accuracy.

关 键 词：胰腺癌 信使RNA 微小RNA 定量PCR 

分 类 号：R-33[医药卫生] R735.9