[1]赵丽,杨洋,温传俊,等.茎-环RT-PCR法定量miRNA-421的引物设计[J].南京师大学报(自然科学版),2012,35(02):83-88.
　Zhao Li,Yang Yang,Wen Chuanjun.Stem-Loop Real-Time Quantitative PCR for Quantification of miRNA-421 by Specific Primers[J].Journal of Nanjing Normal University(Natural Science Edition),2012,35(02):83-88.
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茎-环RT-PCR法定量miRNA-421的引物设计()
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《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

卷:
第35卷
期数:
2012年02期
页码:
83-88
栏目:
生命科学
出版日期:
2012-06-20

文章信息/Info

Title:
Stem-Loop Real-Time Quantitative PCR for Quantification of miRNA-421 by Specific Primers
作者:
赵丽;杨洋;温传俊;
南京师范大学生命科学学院分子细胞生物学研究所，江苏南京210046
Author(s):
Zhao Li; Yang Yang; Wen Chuanjun
Institute of Molecular Cell Biology,School of Life Sciences,Nanjing Normal University,Nanjing 210046,China
关键词:
茎－ 环RT-PCR; miRNA-421; 引物设计
Keywords:
stem-loop RT-PCR; miRNA-421; primers design
分类号:
Q503
摘要:
本研究旨在通过茎-环RT-PCR法,建立有效定量miRNA-421的PCR方法,探讨该方法在定量检测miRNA方面的应用价值.利用miRBase数据库获得miRNA-421的成熟序列.设计3条miRNA-421特异性反转录引物,Q-PCR上游引物及通用下游引物.通过10倍梯度稀释RNA后特异性反转录,RT-PCR分析不同引物互相配对PCR的扩增曲线及熔融曲线,鉴定出特异性好、扩增效率高的引物.为进一步鉴定引物的有效性,过表达miRNA-421,用鉴定的成熟引物定量扩增.利用茎-环RT-PCR法设计、鉴定出miRNA-421引物:421RT7、F19,且这对引物特异性好、扩增效率高.通过设计引物组合,茎-环RT-PCR法能够很好地用于miRNA定量分析,并具有准确、快速、节约成本的优点.
Abstract:
This study was to establish a method of quantitative detecting miRNA-421 by stem-loop RT-PCR，and to evaluate the feasibility of this method in quantitative assay miRNA． miRBase database was used to get the mature sequence of miRNA-421． 6 primers were designed，including 3 primers for miRNA-421 specific reverse transcription，2 forward and 1 general reverse primers for quantitative PCR，respectively． Different RT primers and forward primers were paired in Q-PCR． RNA after 10 times dilution was reverse transcribed and then detected by Q-PCR． Amplification curves and melting curves were analyzed to identify the primers with high specificity and efficiency of amplification． In order to further validate this primer，miRNA-421 was over-expressed in HepG2 and detected by Q-PCR． 2 primers，421RT7 and 421F19 with high specificity and efficiency of amplification were obtained using stem-loop RT-PCR． Stem-loop RT-PCR provided a method that enabled fast，accurate and sensitive detection of miRNA．

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备注/Memo

备注/Memo:
基金项目: 国家自然科学基金( 81171913) 、2008 年江苏省教育厅重大项目( 08KJA520001) ．通讯联系人: 温传俊，副教授，研究方向: 肿瘤的发生、发展及转移的信号调控． E-mail: wenchuanjun@ hotmail． com
更新日期/Last Update: 2013-03-11