【美吉生物】Small&RNA深度测序常见问题
1.如何分离Small RNA？
目前，RNA纯化方法主要包括有机溶剂抽提+乙醇沉淀、硅胶膜离心柱等。由于硅胶膜离心柱只能富集200nt以上的RNA分子，所以并不适用于Small RNA的分离纯化。有机溶剂抽提虽然能够较好的保留Small RNA，但是后期的沉淀步骤非常繁琐。对于Small RNA测序，我们主要采用PAGE胶电泳对Small RNA进行分离。Small RNA的长度为18～30nt，合作伙伴可根据感兴趣的长度进行研究。
2.&采用Illumina进行Small RNA测序时read的长度为多少？
Small RNA的长度为18～30nt，采用Illumina测序平台进行测序，我们推荐的测序长度为35bp，在后期的生物信息分析中，可对序列信息进行修剪，去除接头序列以获得Small RNA序列。
3.有哪些miRNA数据库？
有哪些miRNA预测软件？
5.&有哪些miRNA靶基因预测软件？&
miRNA靶基因预测软件分为第一代预测软件和第二代预测软件，两者的区别是所采用的算法不同，前者大多是从种子互补这一规则出发设计算法，其次才考虑miRNA靶基因跨物种间的保守性；而后者更倾向于采用机器学习方法训练参数进行靶基因预测。现在，多采用第二代target预测方法对miRNA靶基因进行预测。&&&&&&
6.&如何对Small RNA进行注释？注释保守和非保守的Small RNA的方法各不相同。保守的Small RNA是通过与已知的非编码Small RNA数据库比对进行注释，如Non coding RNA
database（http://biobases.ibch.poznan.pl
/ncRNA/）。如果Small RNA拥有常用的专门数据库，还需将待注释的Small RNA与这些数据库比对，注释结果取两者的并集。
剩下的非保守的Small RNA，可根据其序列和结构特征，用相应的软件进行预测，以注释Small RNA。如miRNA的预测过程包括：①
miRNA的前体预测；②
成熟miRNA的预测；③
成熟miRNA靶基因的预测；④
实验验证。
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Micro-RNA: A New Kind of Gene Regulators
Agr icultural &Sciences in &Ch ina&) : 77-80 & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & &January 2006&&
Micro-RNA: A New Kind of Gene Regulators&&WU Dan and HU Lan&&L ivestock &Science and Veterinary &M ed icine I nstitute, Sheny ang Agricultural &Un ivers ity, &Sheny ang &110161, &P.R .China&&Abstract&&A group of small RNA molecules, distinct from but related to siRNA s (small interference RNA s) have been identified in a&variety of organisms . These small RNAs, called microRNAs (miRNA s), are endogenously encoded approximately 20-24 nt&&long single-stranded RNA s. They are generally expressed in a highly tissue- or developmental-stage-specific fashion &and&are post-transcriptional regulator of gene expression in animals and plants. This article summarizes the character, mechanism&&and analysis method about miRNA s . The current view that miRNA s represent a newly discovered, hidden layer of gene&regulation has resulted in high interest among researchers in the discovery of miRNAs, their targets, expression mechanism&of action &and an alysis methods .&&Key words: miRNA s, expression mechanism , analysis method&&& & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & &insignificant, and on the contrary, its abundance and&INSTRUCTION & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & &importance is being gradually recognized by people.&&In the year &1993, Lee (Lee et al. 1993) discovered lin- & & & & & & & & T HE FORMULAT IONAND ST RUCT URE OF&4 in the nematode Caenorhabditis elegans through ge-&netic screens for mutants that lacked the ability to con- & & & & & & & & MAT URE miRNA&&trol the timing of specific cell fate switches during de-&velopment (Lee et al. &1993; Pasquinelli et al. 2000). & & & & & & & & & Based &on &some &r esear ches, &the &matur e &pr oces s &of&After seven years, in the year 2000, Reinhart (Reinhart & & & & & & & & &miRNA (shown in Fig.1) can be generalized as follows:&et &al . 2 000) & &di sc ov er ed & le t- 7 &in th e &n emat o de & & & &( 1) miRNAs &are tr anscribed as parts &of longer mol-&Caenorhabditis elegans. From then on, more and more & & & & & & & & & & &ecules with length up &to &1 000 (2) the RNA s are&researchers and foci dove into this research field. & & & & & & & & & & &processed in the nucleus into 70- 100 nt long hairpin&& &To date, more than &1 100 miRNAs have been identi- & & & & & & & & & &RNAs by Drosha, a dsRNA (double strand RNA)-spe-&fied in invertebrates, vertebrates and plants according & & & & & & & & & (3) the hairpin RNAs are transported&to the miRNA registry database (Griffiths-Jones 2004). & & & & & & & & & to the cytoplasm via a transportin-5 dependent mecha-&There are currently estimated to be -200-255 miRNAs & & & & & & & & & & &nism where they are digested by a second, double-strand&(Lim et al. 2003) present in human transcriptive RNAs & & & & & & & & & &specific ribonuclease called dicer (Grishok et al. 2001).&or transcripteome. In Caenorhabditis elegans, there are & & & & & & & & &Pre-miRNA processing involves Dicer and, presumably,&more than &1000 molecules per cell, with some exceed- & & & & & & & & & &one or more cofactors such as AGL- 1/AGL-2. These&ing 50 000 copies per cell (Lim et al. 2003). These re- & & & & & & & & &factors might then &also be involved in delivery of the&port s &pr oved &that &the &ex istence &of &miRNA s &is &not & & & & & & miRNA to its site of action, i.e., its target mRNA and&&Received 30 March , 2005 Accepted 31 October, 2005&&WU &Dan , M Sc, Tel : &, E-mail : wudan 810225@ Correspondence HU &Lan , Professor&&& & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & & && 2006, CAAS.All rights reserved. Published by Elsevier Ltd.&
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