血清miRNA提取方法的优化
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摘要
利用正交试验法优化血清miRNA的提取方法,为进一步研究血清miRNA作为生物标记物提供方法学基础.本文以hsa-miRNA-223-3p,hsa-miRNA-483-5p和hsa-miRNA-16为检测目标,通过比较Trizol法和Trizol+酚/氯仿法的miRNA提取效率和qPCR检测结果,应用正交试验选择出最优实验方案.结果表明血清miRNA的最优提取方法为:血清用DEPC水4倍稀释,离心条件为4℃,12 000r·min-1,离心时间为10min,Trizol法提取.
To provide the methodological basis for serum miRNA as a biomarker,an orthogonal array design for optimizing miRNA extraction from serum was studied.In this study,RNA extraction efficiency and expression levels of hsa-miRNA-223-3p,hsa-miRNA-483-5pand hsa-miRNA-16 were measured by real-time PCR to make a comparison between single Trizol method,and combination of Trizol and phenol/chloroform method.The results showed that the optimum miRNA extraction method was that serum was diluted 4times with DEPC water,centrifuged at 12 000r·min-1 for 10 min at 4℃ and then used to extract miRNA by Trizol method.
To provide the methodological basis for serum miRNA as a biomarker,an orthogonal array design for optimizing miRNA extraction from serum was studied.In this study,RNA extraction efficiency and expression levels of hsa-miRNA-223-3p,hsa-miRNA-483-5pand hsa-miRNA-16 were measured by real-time PCR to make a comparison between single Trizol method,and combination of Trizol and phenol/chloroform method.The results showed that the optimum miRNA extraction method was that serum was diluted 4times with DEPC water,centrifuged at 12 000r·min-1 for 10 min at 4℃ and then used to extract miRNA by Trizol method.
引文
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[4]Jarry J,Schadendorf D,Greenwood C,et al.The validity of circulating microRNAs in oncology:five years of challenges and contradictions[J].Mol Oncol,2014,8(4):819-829.
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[6]Page K,Guttery D S,Zahra N,et al.Influence of plasma processing on recovery and analysis of circulating nucleic acids[J].PloS One,2013,8(10):e77963.
[7]李春喜,邵云,姜丽娜,等.生物统计学[M].北京:科学出版社,2008.
[8]杨晶,刘嘉琪,王宝昌,等.正交试验优选桑白皮总黄酮的最佳提取工艺[J].辽宁中医杂志,2014,41(2):319-321.
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[10]Zhang T,Wang Q,Zhao D,et al.The oncogenetic role of microRNA-31as a potential biomarker in oesophageal squamous cell carcinoma[J].Clin Sci,2011,121(10):437-447.
[11]Wu C,Li M,Hu C,et al.Clinical significance of serum miR-223,miR-25and miR-375in patients with esophageal squamous cell carcinoma[J].Mol Biol Rep,2014,41(3):1257-1266.
[12]Zhang C,Wang C,Chen X,et al.Expression profile of microRNAs in serum:a fingerprint for esophageal squamous cell carcinoma[J].Clin Chem,2010,56(12):1871-1879.
[13]Cai E H,Gao Y X,Wei Z Z,et al.Serum miR-21expression in human esophageal squamous cell carcinomas[J].Asian Pac J Cancer Prev,2012,13(4):1563-1567.
[14]蒋敏,喻文彬,顾国浩.实时荧光定量PCR检测食管癌血清中miR-21和miR-205的表达及意义[J].广东医学,2014,35(4):535-537.