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    MeRIP-PCR技术服务

    • 簡介
    • 服務特點
    • 樣本要求
    • 結果展示
    • 對差異m6A基因/轉錄本進行qPCR驗證是表觀轉錄研究必不可少的步驟。给我免费播放片高清在线观看生物提供与MeRIP-Seq相對應的MeRIP-PCR服務,保证最优的实验流程与最佳的技术方案。


      圖一. MeRIP-PCR实验流程图。


    • 给我免费播放片高清在线观看丨数谱生物服务特色:

      優化的實驗流程
      特異性強的m6A抗体
      通過嚴格測試的引物

    • 樣本類型新鮮組織、细胞或total RNA
      樣 本 量:≥120μg total RNA或者能获得相应份量的组织细胞,最低浓度不低于50ng/μl
      樣品質量:A260/A280为1.9~2.2;无基因组DNA污染;RNA无降解;
      樣本運輸:RNA样品使用RNAase-Free离心管保存,封口膜封口后干冰运输;组织细胞样品RNAase-Free冻存管保存,干冰或液氮运输;

    • MeRIP-PCR既可对完整的m6A转录本(Intact)进行检测,也可对该转录本的m6A修饰区域(m6A-deposited Fragments)进行检测。



      图二. MeRIP-PCR验证ALKBH5敲除组(shALKBH5)与对照组(shCtrl)之间m6A修饰转录本FOXM1存在明显差异

            (Zhang et al., 2017, Cancer Cell 31, 1–16)



      Normalize each MeRIP RNA fractions’ Ct value to the Input RNA fraction Ct value for the same qPCR Assay (ΔCt) to account for chromatin sample preparation differences. Calculate the % Input for each MeRIP fraction:

      %Input=2(Ct Input-Ct MeRIP)×Fd×100% Here, Fd is Input dilution factor.

      For example, if one- thirtieth of input mRNA and one-fifth of MeRIP mRNA were used for qPCR assays, Fd =5/30 =1/6  

         Fold Enrichment=2[ POS(CT Input-Ct MeRIP)-NEG (CT Input-Ct MeRIP) ]


      sample

      Input-IP

      Input-neg

      IP/neg

      input(Ct)

      IP(Ct)

      %

      input(Ct)

      neg(Ct)

      %

      2FVector C1

      27.515

      32.061

      0.71

      27.515

      NA

      0.01

      61.35

      2F4E C3

      27.287

      32.885

      0.34

      27.287

      NA

      0.01

      34.65

      2FVector C5

      26.652

      25.811

      29.85

      26.652

      NA

      0.01

      4669.09

      2F4E C6

      26.018

      25.145

      30.52

      26.018

      NA

      0.00

      7408.39

      2F4E C3/2FVector C1

       

      0.48

       

      0.85

       

      2F4E C6/2FVector C5

       

      1.02

       

      0.64