不同RNA提取及反转录方法对环状RNA聚合酶链反应的影响  

Effects of different total RNA extraction methods and different reverse transcription primers on circular RNA polymerase chain reaction

作　　者：王建星[1] 聂子元[2] 董金辉[1] 贾新菊[3] WANG Jian-xing;NIE Zi-yuan;DONG Jin-hui;JIA Xin-jun(Department of Hematology, the Second Hospital of Hebei Medical University, Shijiazhuang 050000, China;Department of Otolaryngology, the Second Hospital of Hebei Medical University, Key Laboratory of Hematology in Hebei, Shijiazhuang 050000, China;Department of Endocrine, the Second Hospital of Hebei Medical University, Shijiazhuang 050031, China)

机构地区：[1]河北医科大学第二医院耳鼻喉一科,河北石家庄050000 [2]河北医科大学第二医院血液内科,河北省血液病重点实验室,河北石家庄050000 [3]河北医科大学第一医院内分泌科,河北石家庄050000

出　　处：《河北医科大学学报》2020年第4期431-435,共5页Journal of Hebei Medical University

基　　金：国家自然科学基金(81970216);河北省医学科学研究重点课题(20170560)。

摘　　要：目的比较不同总RNA提取方法及不同反转录引物对喉鳞状细胞癌细胞株Hep2细胞环状RAN扩增的影响,进一步为环状RNA鉴定与扩增提供可靠的实验方法。方法使用Trizol抽提法和miRNeasy Mini试剂盒提取Hep2细胞中的总RNA;Nanodrop 2000对总RNA进行定量及纯度测定;使用随机引物和通用引物分别对总RNA进步反转录;实时定量聚合酶链反应检测Hep2细胞中环状RNA-circHIPK3及circFLNA的表达。结果Trizol抽提法提取的RNA浓度和RNA总量高于miRNeasy Mini试剂盒提取的RNA总浓度结果(P<0.01),但应用miRNeasy Mini试剂盒提取的RNA纯度要高于Trizol抽提法(P<0.05),应用随机引物反转录,能扩增出circHIPK3和circFLNA,而利用通用引物进行反转录,circHIPK3及circFLNA不能被扩增;另外,利用miRNeasy Mini试剂盒法提取总RNA后所扩增出circHIPK3及circFLNA的相对表达量明显高于Trizol抽提法(P<0.01)。结论miRNeasy Mini试剂盒提取总RNA纯度更高,更有利于环状RNA的扩增,利用随机引物进行反转录而非通用引物,可顺利扩增出环状RNA。Objective To investigate the effects of different total RNA extraction methods and different reverse transcription primers on circular RAN amplification Hep2 cell and provide a reliable experimental method for the identification and amplification of circular RNA.Methods Total RNA was extracted from prostate cancer cell line Hep2 cells by using Trizol method and miRNeasy Mini kit protocol.Total RNA was quantified by NanoDorp 2000.Random primer and oligoDT primer were used to reverse transcription of total RNA.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of cicHIPK3 and circFLNA.Results The concentration of RNA extracted by Trizol method was higher than that of RNA extracted by miRNeasy Mini Kit(P<0.01).The purity of RNA extracted by miReasy Mini Kit was higher than that of RNA extracted by Trizol method.The reverse transcription could not amplify the above two circular RNAs,but the universal primers were used for transcription,and both circular RNAs could be amplified;Real-time quantitative polymerase chain result showed that the relative expression of circHIPK3 and circFLNA was higher in miRNeasy Mini method group than in Trizol method(P<0.01).Conclusion The purity of RNA extracted by miRNeasy Mini Kit is higher,which is more conducive to the amplification of circular RNA.

关 键 词：核糖核苷酸类 聚合酶链反应 核苷酸扩增技术 

分 类 号：R342.4[医药卫生—基础医学]