基于SYBR Green Ⅰ的茎-环Real-time PCR定量检测miRNA-7方法的建立  

Stem-loop real-time PCR assay for quantitative detection of miRNA-7 by specific primers using SYBR Green Ⅰ

作　　者：赵娟娟[1,2] 徐华林[1,2] 陶弋婧 郭萌萌[1,2] 周涯[3] 陈超[1,2] 秦娜琳[1,2] 郑静[1,2] 田丹[1,2] 徐林[1,2] 

机构地区：[1]遵义医学院免疫学教研室暨贵州省生物治疗人才基地,贵州遵义563099 [2]贵州省普通高等学校特色药物肿瘤防治特色重点实验室,贵州遵义563099 [3]遵义医学院医学物理学教研室,贵州遵义563099

出　　处：《遵义医学院学报》2015年第6期636-641,共6页Journal of Zunyi Medical University

基　　金：国家自然科学基金资助项目(NO:31370918);教育部"新世纪优秀人才计划"资助项目(NO:NCET-12-0661);贵州省教育厅特色重点实验室建设项目(NO:黔教合KY字[2014]212)

摘　　要：目的本研究旨在利用常规荧光燃料SYBR GreenⅠ,通过茎-环法设计原理,建立有效检测微小RNA-7(miR-7)的Real-time PCR方法。方法利用miRBase数据库获得miR-7的成熟体序列,分别设计1条miR-7特异性反转录引物,以及Real-time PCR上游和下游引物;观察不同退火温度和不同引物浓度对扩增miR-7的Ct值影响,选择最优的退火温度和引物浓度;测定不同稀释度RNA下Real-time PCR扩增miR-7的效果;并利用该方法检测小鼠4个器官中miR-7的灵敏性和特异性。结果在基于SYBR GreenⅠ的茎-环Real-time PCR检测法中,miR-7扩增的最优退火温度为60℃,最优引物浓度为10μmol/L;在不同稀释度RNA下miR-7的Ct值线性关系较好,且在模板RNA低于100 pg的条件下,该方法仍可有效检测miR-7分子;最后有效检测出小鼠4个不同器官中miR-7的差异性表达。结论成功建立基于SYBR GreenⅠ的茎-环Real-time PCR定量检测miRNA-7的方法,可为后续研究miR-7的生物学功能提供了重要工作基础。Objective To establish an effective method of Real-time PCR quantitative detection of microRNA-7（miR-7）,based on the principles of stem-loop theory using conventional fluorochrome SYBR GreenⅠ.Methods Mature sequences of miR-7 were obtained from miRBase database,and then 3 primers were designed,including a specific reverse transcription primer,as well as one forward and one reverse primer for Real-time PCR.Next,different annealing temperatures and distinct concentrations of primer were performed for the analysis of optimal Ct values of miR-7 in Real-time PCR assay.Furthermore,different concentrations of RNA were used for the detection of miR-7 expression level in the condition of optimal annealing temperature and primer concentration.Finally,the distinct expression level of miR-7 in murine four different organs was analyzed.Results In stem-loop Real-time PCR assay,the optimal annealing temperature and primer concentration for the amplification of miR-7 were 60 ℃ and 10 μmol/l,respectively.Moreover,Ct values of miR-7 showed good linear relationship detection,and the level of miR-7 could be also detected in samples with 100 pg RNAs.Finally,the distinct level of miR-7 expression in murine four different organs could be determined by stem-loop Real-time PCR assay.Conclusion The stem-loop Real-time PCR using SYBR Green Ⅰcould be well used for the quantitative analysis of miR-7,which provided the bases for the successive research work on the biological function of miR-7.

关 键 词：茎-环Real-time PCR法 SYBR GreenⅠ 微小RNA-7 表达 

分 类 号：R392-33[医药卫生—免疫学]