[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.
点击复制

茎-环RT-PCR法定量miRNA-421的引物设计()
分享到:

《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 LiYang YangWen Chuanjun
Institute of Molecular Cell Biology,School of Life Sciences,Nanjing Normal University,Nanjing 210046,China
关键词:
茎- 环RT-PCRmiRNA-421引物设计
Keywords:
stem-loop RT-PCRmiRNA-421primers 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.

参考文献/References:

[1] Buckingham M. Myogenic progenitor cells and skeletal myogenesis in vertebrates[J]. Curr Opin Genet Dev,2006,16( 5) : 525-532.
[2] Bartel D P. MicroRNAs: target recognition and regulatory functions[J]. Cell,2009,136( 2) : 215-233.
[3] Bartel D P. MicroRNAs: genomics,biogenesis,mechanism,and function[J]. Cell,2004, 116( 2) : 281-297.
[4] Bentwich I,Avniel A,Karov Y,et al. Identification of hundreds of conserved and nonconserved human microRNAs[J]. Nat Genet,2005,37( 7) : 766-770.
[5] Lewis B P,Burge C B,Bartel D P. Conserved seed pairing,often flanked by adenosines,indicates that thousands of human genes are microRNA targets[J]. Cell,2005,120( 1) : 15-20.
[6] Lim L P,Glasner M E,Yekta S,et al. Vertebrate microRNA genes[J]. Science,2003,299( 5612) : 1 540.
[7] Krichevsky A M,King K S,Donahue C P,et al. A microRNA array reveals extensive regulation of microRNAs during brain development[J]. RNA,2003,9( 10) : 1 274-1 281.
[8] Ambros V,Lee R C. Identification of microRNAs and other tiny noncoding RNAs by cDNA cloning[J]. Mol Biol,2004, 265: 131-158.
[9] Shi R,Chiang V L. Facile means for quantifying microRNA expression by real-time PCR[J]. Biotechniques,2005,39( 4) : 519-525.
[10] Raymond C K,Roberts B S,Garrett-Engele P,et al. Simple,quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs[J]. RNA,2005,11( 11) : 1 737-1 744.
[11] Chen C,Ridzon D A,Broomer A J,et al. Real-time quantification of microRNAs by stem-loop RT-PCR[J]. Nucleic Acids Res,2005,33( 20) : e179.
[12] Jiang Z,Guo J,Xiao B,et al. Increased expression of miR-421 in human gastric carcinoma and its clinical association[J]. J Gastroenterol,2010,45( 1) : 17-23.
[13] Hao J,Zhang S,Zhou Y,et al. MicroRNA 421 suppresses DPC4 /Smad4 in pancreatic cancer[J]. Biochem Biophys Res Commun,2011,406( 4) : 552-557.[14] Hu H,Du L,Nagabayashi G,et al. ATM is down-regulated by N-Myc-regulated microRNA-421[J]. Proc Natl Acad Sci USA,2010,107( 4) : . 1 506-1 511.[15] Lee R C,Feinbaum R L,Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J]. Cell,1993, 75( 5) : 843-854.
[16] He L,Thomson J M,Hemann M T,et al. A microRNA polycistron as a potential human oncogene[J]. Nature,2005,435 ( 7043) : 828-833.
[17] Mraz M,Pospisilova S,Malinova K,et al. MicroRNAs in chronic lymphocytic leukemia pathogenesis and disease subtypes [J]. Leuk Lymphoma,2009,50( 3) : 506-509.
[18] Thum T,Galuppo P,Wolf C,et al. MicroRNAs in the human heart: a clue to fetal gene reprogramming in heart failure [J]. Circulation,2007,116( 3) : 258-267.
[19] van Rooij E,Sutherland L B,Qi X,et al. Control of stress-dependent cardiac growth and gene expression by a microRNA [J]. Science,2007,316( 5824) : 575-579.
[20] Mencia A,Modamio H S,Redshaw N,et al. Mutations in the seed region of human miR-96 are responsible for nonsyndromic progressive hearing loss[J]. Nat Genet,2009,41( 5) : 609-613.
[21] Hughes A E,Bradley D T,Campbell M,et al. Mutation altering the miR-184 seed region causes familial keratoconus with cataract[J]. Am J Hum Genet,2011,89( 5) : 628-633.
[22] de Pontual L,Yao E,Callier P,et al. Germline deletion of the miR-17 approximately 92 cluster causes skeletal and growth defects in humans[J]. Nat Genet,2011,43( 10) : 1 026-1 030.
[23] Ma L,Young J,Prabhala H,et al. miR-9,a MYC/MYCN-activated microRNA,regulates E-cadherin and cancer metastasis [J]. Nat Cell Biol,2010,12( 3) : 247-256.
[24] Tang F,Hajkova P,Barton S C,et al. MicroRNA expression profiling of single whole embryonic stem cells[J]. Nucleic Acids Res,2006,34( 2) : e9.

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