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    LNCRNA研究策略

                  

     

    一、 lncRNA筛?。?/span>

    yy智勇三张牌 www.hvfh.net  

    • 1. 通过lncRNA芯片或RNA测序等方法对多对疾病模型和对照样本组织进行lncRNA表达谱分析;
    •  
    • 2.通过生物信息学的方法筛选出具有表达差异的lncRNA,构建共表达网络,预测lncRNA的靶基因;
    •  
    • 3.通过PCR或NorthernBlot技术对候选lncRNA验证,确定其表达差异。

     


    二、lncRNA确定:

     

    • 1.通过5'RACE获取lncRNA5'全长,3'RACE获取lncRNA3'全长,最终拿到完整的lncRNA序列。


    三、表达分析:

     

    • 1.细胞水平表达:在细胞水平进行检测表达差异。
      
    • 2.组织分布:检测不同组织、不同阶段表达特性。
      
    • 3.表达水平动力学变化:比较不同处理条件下,如药物处理、诱导处理下,表达水平差异。
       


    四、 功能研究:

     

    • 1.功能获得性研究:构建lncRNA过表达载体:原则上是将全长lncRNA定向克隆到表达载体上实现lncRNA的过表达。然而有些lncRNA很大或全长尚未分离,这时将视lncRNA在 基因组上 的定位采取不同的研究策略。
    •  
       
    • 2.功能缺失性研究:可通过siRNA、shRNA、反义核酸等方法沉默lncRNA,干预lncRNA后检测其对疾病相关基因表达的影响和对细胞表型如增值、凋亡、侵袭、转移等的影响;
    •  
    • 3.采用RNApulldown、RNA-RIP(RNABindingProteinImmunoprecipitation)、ChIRP-seq(ChromatinIsolationbyRNAPurification)等方法检测与lncRNA结合的DNA、RNA、蛋白质。
    •  
    • 4.采用lncRNA芯片分析技术结合mRNA对lncRNA功能进行预测,研究lncRNAtrans和cis作用机制。
    •  
    • 5.采用配体指数级富集系统进化技术(systematicevolutionofligandsbyexponentialenrichment,SELEX),设计一种RNA配体,与癌症相关的lincRNA结合达到抑制肿瘤细胞的生长和转移。
    •  
    • 6.采用快速预测RNA与蛋白质相互作用与结构域(catRAPID)在线算法来预测RNA与蛋白质的相互作用,该算法根据RNA和蛋白质的二级结构、氢键和分子作用力来评估它们之间的相互作用的倾向。
    •  
    • 7.采用非编码RNA沉默和定位分析(c-KLAN)技术对lncRNA进行功能缺失(Loss-of-function)研究和细胞定位,该技术是在基于核糖核酸内切酶制备的小干扰RNA(esiRNA)和荧光原位杂交(FISH)2种现有的研究方法的基础上建立起来的。


    五、 表达调控:将lncRNA表达与其他领域相结合,解释lncRNA调控机理。

     

    • 1.DNA甲基化:可通过检测相应基因甲基化差异与lncRNA结合分析。
    •  
    • 2.转录因子:研究lncRNA与转录因子的调控机制。
    •  
    • 3.染色质重塑:lncRNA表观调控。

     
    六、LncRNA研究案例:

     

     

    七、lncRNA的研究背景及其与疾病的关系

     

     

    相关链接

  • 【专题】河北省无偿献血领导小组会议报道 2019-05-05
  • 我写文章不是为了别人的赞许,是为了讨论问题,让人有思考的价值,就像你网名一样,探寻真理。我并非就全盘赞成市场经济,只是在讨论它的合理性,在文中也提问,“既然我们 2019-05-05
  • 机构改革是一场政治考验 2019-05-03
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  • 危急时刻,她把生的希望留给学生 2019-04-19
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