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LNCRNA研究策略

              

 

一、 lncRNA篩?。?/span>

广东快乐十分直播开奖 www.netjwr.com.cn  

  • 1. 通過lncRNA芯片或RNA測序等方法對多對疾病模型和對照樣本組織進行lncRNA表達譜分析;
  •  
  • 2.通過生物信息學的方法篩選出具有表達差異的lncRNA,構建共表達網絡,預測lncRNA的靶基因;
  •  
  • 3.通過PCR或NorthernBlot技術對候選lncRNA驗證,確定其表達差異。

 


二、lncRNA確定:

 

  • 1.通過5'RACE獲取lncRNA5'全長,3'RACE獲取lncRNA3'全長,最終拿到完整的lncRNA序列。


三、表達分析:

 

  • 1.細胞水平表達:在細胞水平進行檢測表達差異。
  
  • 2.組織分布:檢測不同組織、不同階段表達特性。
  
  • 3.表達水平動力學變化:比較不同處理條件下,如藥物處理、誘導處理下,表達水平差異。
     


四、 功能研究:

 

  • 1.功能獲得性研究:構建lncRNA過表達載體:原則上是將全長lncRNA定向克隆到表達載體上實現lncRNA的過表達。然而有些lncRNA很大或全長尚未分離,這時將視lncRNA在 基因組上 的定位采取不同的研究策略。
  •  
     
  • 2.功能缺失性研究:可通過siRNA、shRNA、反義核酸等方法沉默lncRNA,干預lncRNA后檢測其對疾病相關基因表達的影響和對細胞表型如增值、凋亡、侵襲、轉移等的影響;
  •  
  • 3.采用RNApulldown、RNA-RIP(RNABindingProteinImmunoprecipitation)、ChIRP-seq(ChromatinIsolationbyRNAPurification)等方法檢測與lncRNA結合的DNA、RNA、蛋白質。
  •  
  • 4.采用lncRNA芯片分析技術結合mRNA對lncRNA功能進行預測,研究lncRNAtrans和cis作用機制。
  •  
  • 5.采用配體指數級富集系統進化技術(systematicevolutionofligandsbyexponentialenrichment,SELEX),設計一種RNA配體,與癌癥相關的lincRNA結合達到抑制腫瘤細胞的生長和轉移。
  •  
  • 6.采用快速預測RNA與蛋白質相互作用與結構域(catRAPID)在線算法來預測RNA與蛋白質的相互作用,該算法根據RNA和蛋白質的二級結構、氫鍵和分子作用力來評估它們之間的相互作用的傾向。
  •  
  • 7.采用非編碼RNA沉默和定位分析(c-KLAN)技術對lncRNA進行功能缺失(Loss-of-function)研究和細胞定位,該技術是在基于核糖核酸內切酶制備的小干擾RNA(esiRNA)和熒光原位雜交(FISH)2種現有的研究方法的基礎上建立起來的。


五、 表達調控:將lncRNA表達與其他領域相結合,解釋lncRNA調控機理。

 

  • 1.DNA甲基化:可通過檢測相應基因甲基化差異與lncRNA結合分析。
  •  
  • 2.轉錄因子:研究lncRNA與轉錄因子的調控機制。
  •  
  • 3.染色質重塑:lncRNA表觀調控。

 
六、LncRNA研究案例:

 

 

七、lncRNA的研究背景及其與疾病的關系

 

 

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