基于实时荧光定量反转录PCR技术的动物miRNA表达分析策略

›› 2013, Vol. 40 ›› Issue (9): 109-113.

基于实时荧光定量反转录PCR技术的动物miRNA表达分析策略

宋嘉哲, 孙福伯, 庄开歌, 薛恺

大连医科大学, 辽宁大连 116044

收稿日期:2013-05-06 出版日期:2013-09-20 发布日期:2013-09-18
通讯作者: 薛恺 E-mail:nkx123xuekai@sina.com
作者简介:宋嘉哲(1981-),女,辽宁人,博士,讲师,研究方向:生物化学与分子生物学。
基金资助:
国家自然科学基金(31101717)。

Real-time Quantitative RT-PCR Analysis of Animal miRNA Expression

SONG Jia-zhe, SUN Fu-bo, ZHUANG Kai-ge, XUE Kai

Dalian Medical University, Dalian 116044, China

Received:2013-05-06 Online:2013-09-20 Published:2013-09-18

摘要/Abstract

摘要： 在人体内有约60%的蛋白质编码基因受到到miRNA调控。近年来,miRNA参与调控的与动物生长相关的细胞周期、发育、分化和新陈代谢等多种生物学过程也越来越受到关注。目前针对miRNA表达检测的常用方法有很多,其中基于反转录PCR(RT-PCR)的放大检测技术是应用范围最广,实施手段最灵活的一种研究策略。主要包括miRNA的全定量PCR法(miR-Q)、基于茎环引物的实时荧光定量PCR法、Poly(A) 加尾性通用反转录法、miRNA表达谱的高通量RT-PCR分析和miRNA扩增谱系(mRAP)法。作者对这几种基于实时荧光定量RT-PCR技术的动物miRNA表达分析策略分别进行了介绍,对其原理、方法和应用范畴进行了概括性总结。

关键词: 动物miRNA; 实时荧光定量PCR; miRNA的全定量PCR; 茎-环引物; miRNA扩增谱系

Abstract: About 60% of human protein-coding genes are regulated by miRNA. These genes produce different proteins, which participant in many biological processes. The functions of these proteins are involved in cell cycle,development,differentiation and metabolism. Nowadays, researchers can detect the expressions of miRNA by various approaches. However, the RT-PCR-based amplifying techniques are the most flexible methods, which are applied in extensive fields. The RT-PCR-based amplifying techniques mainly include miR-Q RT-PCR for miRNA quantification, end-point stem-loop Real-time RT-PCR for miRNA quantification, Poly(A)-tailed universal reverse transcription, multiplexing RT-PCR for high-throughput miRNA profiling and miRNA amplification profiling (mRAP). Here we introduce these important Real-time PCR methods for detecting miRNA, respectively. And the theory, strategy, application and limitation of each method are summarized synoptically.

Key words: animal miRNA; Real-time PCR; miRNA quantitative RT-PCR; stem-loop primer; miRNA amplification profile

中图分类号:

宋嘉哲, 孙福伯, 庄开歌, 薛恺. 基于实时荧光定量反转录PCR技术的动物miRNA表达分析策略[J]. , 2013, 40(9): 109-113.

SONG Jia-zhe, SUN Fu-bo, ZHUANG Kai-ge, XUE Kai. Real-time Quantitative RT-PCR Analysis of Animal miRNA Expression[J]. , 2013, 40(9): 109-113.

参考文献

1 李虹仪, 张永亮, 习欠云, 等. 猪miRNA的研究进展[J]. 中国畜牧兽医, 2010, 37(1):80~84.
2 Chen C, Ridzon D A, Broomer A J, et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res, 2005, 33:e179.
3 Friedman R C, Farh K K, Burge C B, et al. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res, 2009, 19:92~105.
4 Fu H J, Zhu J, Yang M, et al. A novel method to monitor the expression of microRNAs. Mol Biotechnol,2006, 32:197~204.
5 Jin P, Alisch R S, Warren S T. RNA and microRNAs in fragile X mental retardation. Nat Cell Biol,2004, 6:1048~1053.
6 Khraiwesh B, Arif M A, Seumel G I, et al. Transcriptional control of gene expression by microRNAs. Cell,2010, 140, 111~122.
7 Landthaler M, Yalcin A, Tuschl T. The human DiGeorge syndrome critical region gene 8 and its D. melanogaster homolog are required for miRNA biogenesis. Curr Biol,2004, 14:2162~2167.
8 Lao K, Xu N L, Yeung V, et al. Multiplexing RT-PCR for the detection of multiple miRNA species in small samples. Biochem Biophys Res Commun,2006, 343:85~89.
9 Liang R Q, Li W, Li Y, et al. An oligonucleotide microarray for microRNA expression analysis based on labeling RNA with quantum dot and nanogold probe. Nucleic Acids Res,2005, 33:e17.
10 Lu J, Getz G, Miska E A, et al. MicroRNA expression profiles classify human cancers. Nature, 2005, 435:834~838.
11 Mano H, Takada S. mRAP, a sensitive method for determination of microRNA expression profiles. Methods, 2007, 43:118~122.
12 Poy M N, Eliasson L, Krutzfeldt J, et al. A pancreatic islet-specific microRNA regulates insulin secretion. Nature, 2004, 432:226~230.
13 Ro S, Park C, Jin J, et al. A PCR-based method for detection and quantification of small RNAs. Biochem Biophys Res Commun, 2006, 351: 756~763.
14 Takada S, Berezikov E, Yamashita Y, et al. Mouse microRNA profiles determined with a new and sensitive cloning method. Nucleic Acids Res,2006, 34:e115.
15 Takada S, Mano H. Profiling of microRNA expression by mRAP. Nat Protoc,2007,2:3136~3145.
16 Volinia S, Calin G A, Liu C G, et al. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci USA, 2006, 103:2257~2261.
17 Wark A W, Lee H J, Corn R M. Multiplexed detection methods for profiling microRNA expression in biological samples. Angew Chem Int Ed Engl, 2008, 47:644~652.
18 Wu X M, Liu M Y, Ge X X, et al. Stage and tissue-specific modulation of ten conserved miRNAs and their targets during somatic embryogenesis of Valencia sweet orange. Planta, 2011,233(3):495~505.

[1]	麻宝艺, 盛陈艳, 刘宏莹, 马青霞, 汪婷婷, 李健明, 时坤, 杜锐, 冷雪. BVDV 1型和2型TaqMan双重实时荧光定量PCR检测方法的建立[J]. 中国畜牧兽医, 2022, 49(6): 2326-2335.
[2]	吕晓楠, 徐立蒲, 张文, 王娜, 曹欢, 王小亮, 王姝, 王静波. 感染鲤浮肿病毒镜鲤的组织病理变化及病毒分布规律研究[J]. 中国畜牧兽医, 2022, 49(3): 1077-1084.
[3]	张军芳, 孙建富, 孙斌, 唐琳, 王英, 王恩泽, 崔岩, 李强, 严昌国, 李香子. 延边牛PPARγ基因的克隆及生物信息学分析[J]. 中国畜牧兽医, 2021, 48(8): 2695-2704.
[4]	苗曼宁, 郭益文, 胡德宝, 曾雨晗, 李新, 张林林, 丁向彬, 郭宏. MSTN基因编辑牛肌肉转录组测序及生物信息学分析[J]. 中国畜牧兽医, 2021, 48(7): 2271-2281.
[5]	李恒, 字向东. 牦牛跨膜附睾蛋白1基因的克隆及其在卵丘卵母细胞复合体中的表达分析[J]. 中国畜牧兽医, 2021, 48(4): 1284-1293.
[6]	李建梅, 季正剑, 刘梅, 沈欣悦, 程旭, 俞燕, 戴亚斌. 藏鸡和隐性白羽鸡感染柔嫩艾美耳球虫后免疫相关基因的表达变化分析[J]. 中国畜牧兽医, 2021, 48(4): 1440-1448.
[7]	陈磊, 袁枫, 贺三刚, 玛依拉, 李文蓉. 绵羊FGF10基因克隆、序列分析及其在毛囊发育中的表达分析[J]. 中国畜牧兽医, 2021, 48(2): 425-432.
[8]	戴政列, 朱静静, 陈振宇, 周晓龙, 汪涵, 李向臣, 赵阿勇, 杨松柏. 猪SENP1基因克隆、生物信息学分析与表达研究[J]. 中国畜牧兽医, 2021, 48(12): 4382-4390.
[9]	姜懿轩, 王亚玲, 邢珊珊, 宋国华, 许会芬, 李明. 新西兰白兔MSTN基因克隆、生物信息学分析及表达谱构建[J]. 中国畜牧兽医, 2021, 48(10): 3501-3511.
[10]	李志强, 陶晨雨, 魏奥龙, 贾青. 猪性控精子纯度实时荧光定量PCR检测方法的建立[J]. 中国畜牧兽医, 2021, 48(10): 3682-3690.
[11]	王征帆, 朱利塞, 王娟, 李祎云, 向蕊, 应碧云, 王贵平, 贾爱卿, 白挨泉. PDCoV与TGEV双重实时荧光定量PCR检测方法的建立及初步应用[J]. 中国畜牧兽医, 2021, 48(10): 3855-3863.
[12]	李安奇, 蒋蕾, 苏晓彤, 昝林森, 王洪宝. 秦川牛ACTC1基因的表达特性分析[J]. 中国畜牧兽医, 2020, 47(8): 2359-2367.
[13]	李京徽, 邢思远, 王希彩, 李庆贺, 赵桂苹, 张永宏, 文杰, 刘冉冉. 鸡肌内脂肪沉积相关候选基因筛选[J]. 中国畜牧兽医, 2020, 47(6): 1828-1836.
[14]	姜徽, 刘攀, 李媛媛, 刘奎国, 黄锡霞, 刘江卫, 胥磊, 杜建文, 桑扎根, 杨雪梅, 赵番番, 王丹. 新疆褐牛乳房炎抗性相关基因的表达及生物信息学分析[J]. 中国畜牧兽医, 2020, 47(1): 90-97.
[15]	张继, 易鸣, 程朝友, 程均华, 付正仙, 杨德文, 吴雪, 邱淦远, 刘若余. 威宁绵羊SOCS7和GFAP基因组织表达研究[J]. 《中国畜牧兽医》, 2019, 46(7): 1899-1906.