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RNP-IP (Modified Method)-Getting Majority of RNA from RNA Binding Protein in Cytoplasm
核糖核蛋白-免疫共沉淀(改进方法)—从细胞质所包含的RNA结合蛋白中分离大部分的RNA   

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Abstract

Post-transcriptional regulation of gene expression is a ribonucleoprotein (RNP)-driven process, which involves RNA binding proteins (RBPs) and noncoding RNAs that regulate splicing, nuclear export, subcellular localization, mRNA stability and translation. mRNAs encoding proteins that function in a particular cell process or pathway can be found within a unique mRNP complex, which consists of mRNA and RNP. This provides valuable information regarding not only known components of a particular process or pathway, but importantly, leads to the identification of novel components representing potential therapeutic targets and biomarkers. In addition to those targets identified by pathway expansion, the specific RBPs (RNA binding proteina) regulating RNA functions may be potential therapeutic targets in their own right. RNP-IP is a technology that allows the isolation and identification of mRNAs, microRNAs and protein components of RNP complexes from cell extracts using antibodies to RBPs. Once purified, the RNAs present in the complex are analyzed to identify the target mRNAs using various molecular biology tools such as RT-PCR, gene expression analysis based on microarray technology (chip analysis), or sequencing. Using this modified method will get more RNA existing in cytoplasm. This method does not require a pre-clear step and getting the supernatant for western blot is different from the original method.

Materials and Reagents

  1. Normal rabbit IgG
  2. RIP-certified antibody (NBL, catalog number depends on what do you want to target)
  3. NaCl
  4. MgCl2
  5. Nonidet P40
  6. NaoAc
  7. Protein A beads (GE Healthcare Dharmacon, catalog number: 17-0780-01 ) or Protein G beads (Thermo Fisher Scientific, catalog number: 22852 )
  8. Ethanol (molecular biology grade)
  9. 2-Propanol (Molecular Biology)
  10. Nuclease-free PBS
  11. Nuclease-free water
  12. Isotype control IgG (if necessary)
  13. Digitornin
  14. Aprotinin (final concentration 10 μg/ml)
  15. Leupeptin (5 μg/ml)
  16. Phenylmethylsulfonyl fluoride (PMSF) (final concentration 0.5 mM)
  17. RNase inhibitor (Life Technologies, Invitrogen™, catalog number: 10777-019 )
  18. Dithiothreitol (DTT) (reducing agent)
  19. Lysis buffer (see Recipes)
  20. Wash buffer (NT2) (see Recipes)
  21. Precaution: Additional buffer preparation (see Recipes)

Equipment

  1. Microcentrifuge capable of 15,000 x g
  2. Microcentrifuge tube (1.5 ml or 2 ml) (nuclease-free) (recommendation; use low-adhesion tube for RIP-Assay)
  3. Centrifuge capable of 2,000 x g
  4. Centrifuge tube (15 ml or 50 ml)
  5. Pipette (5 ml, 10 ml, 25 ml) (nuclease-free)
  6. Pipette tip (10 μl, 20-100 μl , 200 μl , and 1,000 μl) (nuclease-free) (recommendation; use low-adhesion pipette tip for RIP-Assay)
  7. Ultra low temperature freezer (-80 °C)
  8. Freezer (below -20 °C)
  9. End-over-end rotator
  10. Vortex mixer
  11. Gloves

Procedure

  1. Preparation of antibody-immobilized protein A or protein G agarose beads.
    Day 1
    1. The following components in DEPEC treated water are final concentrations.
      50 mM Tris (pH 7.5)
      50 mM NaCl
      1 mM MgCl2
      0.05% Nonidet P40
    2. Wash beads with NT2 buffer. If you have 4 samples, you need to take 100 μl beads to each Eppendorf tube, total 4 tubes. 3,600 rpm 1 min at 4 °C, remove supernatant. Wash 3 times with 200 μl NT2 buffer.
    3. Add 30 μg target antibody (such as HuR 1 μg/μl from NBL) to each tube.
    4. Add 320 μl NT2 buffer to each tube. Overnight rotate at 4 °C.
    Day 2
    1. Centrifuge the tubes (Ab+ beads) 5,000 x g 5 min at 4 °C. Discard supernatant wash twice with freshly made NT2 buffer 1 ml. After last wash, temporarily keep supernatant and put on ice until cell lysate is ready.

  2. Lysis of mammalian cells
    1. Prepare cell pellet:
      1. Detach the cells from the culture dish by pipetting or using a cell scraper, if necessary. Collect the cell suspension into centrifuge tube.
      2. Centrifuge the cell suspension at 300 x g for 5 min at 4 °C to pellet the cells. Carefully remove and discard the supernatant.
      3. Wash the cells by resuspending the cell pellet with ice-cold PBS.
      4. Centrifuge the cell suspension at 300 x g for 5 min at 4 °C to pellet the cells. Carefully remove and discard the supernatant.
      5. Wash the cells once again using steps 3-4.
      6. Wash the cells by resuspending the cell pellet with ice-cold nuclease-free PBS.
      7. Centrifuge the cell suspension at 300 x g for 5 min at 4 °C to pellet the cells. Carefully remove and discard the supernatant.
      8. Wash the cells by resuspending the cell pellet with ice-cold nuclease-free PBS.
      9. Aliquot the cell suspension to each new microcentrifuge tube.
      10. Centrifuge the cell suspension at 300 x g for 5 min at 4 °C to pellet the cells. Carefully remove and discard the supernatant.
    2. Lysis cell pellet
      1. Make RSB buffer in DEPEC treated water with final following concentrations.
        10 mM Tris (pH 7.5)
        100 mM NaCl
        2.5 mM MgCl2
        Then add other supplements in per ml RSB buffer.
        10 μl digitornin/ml buffer (Digitornin is dissolved in ethanol 4 mg/ml freshly), 3 μl RNase inhibitor/ml buffer, 40 μl proteinase inhibitor cocktail/ml.
      2. Add 200 μl of RSB buffer to each cell pellet. On ice 2 min. Centrifuge 4,400 rpm 4  °C, 8 min.
      3. Make 753 μl master mix for each tube (Ab + beads). For 4 tubes, you can make 4.5 times more.
        700 μl NT2 buffer
        10 μl 0.1 M DTT
        10 μl RNase inhibitor
        33 μl 0.5 M EDTA (pH 8)
      4. Then add each supernatant of cell lysate to tube (Ab+ beads). Rotate at 4 °C 2 h.
      5. Take 20 μl for western blot from the mixture. The centrifuge the tube at 5,000 x g 4 °C 2 min.
      6. Discard supernatant. Wash pellet with 1 ml NT2 buffer twice at 5,000 x g 4 °C 2 min.
      7. Further process the mixture to get mRNAs. Make DNAase master mix.
        Now we have 4 tubes, so make 4.5 times master mix.
        100 μl of NT2 buffer x 4.5=450 μl
        5 μl of DNAase (2U/μl) x 4.5=22.5 μl
        Add 105 μl of DNAase master mix to each tube and 37 °C, 10 min incubation.
      8. Then add 1ml NT2 buffer centrifuge 5,000 x g 5 min 4 °C. Remove supernatant.
      9. Add proteinase K (20 mg/μl) master mix 103 μl to each tube. 55 °C, 15 min plus mixing.
        Master mix: 2.5 μl proteinase K x 4.5=11.25 μl
        0.5 μl 20% SDS x 4.5=2.25 μl
        100 μl NT2 buffer x 4.5=450 μl
      10. Centrifuge 5,000 x g 4 °C 5 min.
      11. Collect 100 μl supernatant to a new Eppendorf tube. Add 200 μl of NT2 buffer to old tube, centrifuge 5,000 x g 4 °C, 5 min again. Collect 200 μl of supernatant and pool to the new tube, which becomes 300 μl of supernatant.
      12. Add equal volume of acid phenol/chloroform to above supernatant.
        Vertex 1 min, then centrifuge 1 min RT, 13k rpm.
      13. Collect about 250 μl of aqueous phase (top phase)
        Add: 25 μl of 3 M NaoAc (pH 5.5)
        625 μl of 100% ethanol
        3 μl of glycogen blue
        Mix well and put it at -20 °C or below for 20 min (or for overnight, if necessary).
      14. Centrifuge the tube at 12,000 x g for 10 min at 4 °C, then aspirate the supernatant carefully.
      15. Rinse with 1 ml 70% Ethanol, 13,000 x g 2 min at 4 °C. Discard supernatant. Invert the tube, air dry 5 min. The pellet is mRNAs.

Recipes

  1. Lysis buffer
    Add appropriate concentrations of protease inhibitors, RNase inhibitor, and DTT to lysis buffer just before use. Lysis buffer containing these reagents is described as lysis buffer (+) in the following protocols. The optimal concentration of each reagent for RIP-Assay is shown as follows.
    Make RSB buffer in DEPEC treated water with final concentrations as follows:
    10 mM Tris (pH 7.5)
    100 mM NaCl
    2.5 mM MgCl2
    Then add other supplements in per ml RSB buffer.
    10 μl digitornin/ml buffer (digitornin is dissolved in ethanol 4 mg/ml freshly), 3 μl RNase inhibitor/ml buffer, 40 μl proteinase inhibitor cocktail/ml.
  2. Wash buffer
    Add final 1.5 mM concentration of DTT to wash buffer just before use. Wash buffer containing DTT is described as wash buffer (+) in the following protocols.
    Make washing buffer in DEPEC treated water with final concentrations as follows:
    50 mM Tris (pH 7.5)
    50 mM NaCl
    1 mM MgCl2
    0.05% Nonidet P40
  3. Precaution: Additional buffer preparation
    In some cases, both the lysis buffer (+) and wash buffer (+) may require the addition of appropriate volumes of high-salt solution (in these cases, add 30 μl of high-salt solution to each ml of lysis buffer and wash buffer).

Acknowledgments

This protocol was adapted from the NBL RIP-Assay Kit (see Reference 1).

References

  1. Protocol from NBL RIP-Assay Kit (NBL, catalog number: RN1001).

简介

基因表达的转录后调节是核糖核蛋白(RNP)驱动的过程,其涉及调节剪接,核出口,亚细胞定位,mRNA稳定性和翻译的RNA结合蛋白(RBP)和非编码RNA。编码在特定细胞过程或途径中起作用的蛋白质的mRNA可在由mRNA和RNP组成的独特mRNP复合物内发现。这不仅提供了关于特定过程或途径的已知组分的有价值信息,而且重要的是导致鉴定代表潜在治疗靶标和生物标志物的新组分。除了通过途径扩增鉴定的那些靶标之外,调节RNA功能的特异性RBP(RNA结合蛋白)可以是它们自己的潜在治疗靶标。 RNP-IP是一种允许使用针对RBP的抗体从细胞提取物中分离和鉴定RNP复合物的mRNA,微小RNA和蛋白质组分的技术。一旦纯化,使用各种分子生物学工具例如RT-PCR,基于微阵列技术(芯片分析)或测序的基因表达分析,分析复合物中存在的RNA以鉴定靶mRNA。使用这种修改的方法将获得更多的RNA存在于细胞质中。该方法不需要预清除步骤,并且获得用于western印迹的上清液不同于原始方法。

材料和试剂

  1. 正常兔IgG
  2. RIP认证的抗体(NBL,目录号取决于您要定位的目标)
  3. NaCl
  4. MgCl 2
  5. Nonidet P40
  6. NaoAc
  7. 蛋白A珠(GE Healthcare Dharmacon,目录号:17-0780-01)或Protein G珠(Thermo Fisher Scientific,目录号:22852)
  8. 乙醇(分子生物学级)
  9. 2-丙醇(分子生物学)
  10. 无核酸酶的PBS
  11. 无核酸酶水溶液
  12. 同种型对照IgG(如有必要)
  13. Digitornin
  14. 抑肽酶(终浓度10μg/ml)
  15. 亮肽素(5μg/ml)
  16. 苯甲基磺酰氟(PMSF)(终浓度0.5mM)
  17. RNase抑制剂(Life Technologies,Invitrogen TM,目录号:10777-019)
  18. 二硫苏糖醇(DTT)(还原剂)
  19. 裂解缓冲液(见配方)
  20. 洗涤缓冲液(NT2)(参见配方)
  21. 注意:附加缓冲液准备(参见配方)

设备

  1. 微量离心机能够处理15,000个电子邮件
  2. 微量离心管(1.5ml或2ml)(不含核酸酶)(建议;使用低附着管RIP-测定)
  3. 离心机能够2,000 x g
  4. 离心管(15ml或50ml)
  5. 移液管(5ml,10ml,25ml)(不含核酸酶)
  6. 移液管吸头(10μl,20-100μl,200μl和1,000μl)(不含核酸酶)(推荐使用RIP-Assay的低附着力吸头)
  7. 超低温冰箱(-80℃)
  8. 冰柜(-20°C以下)
  9. 端到端旋转器
  10. 涡流搅拌器
  11. 手套

程序

  1. 抗体固定的蛋白A或蛋白G琼脂糖珠的制备 第1天
    1. 在DEPEC处理的水中的以下组分是最终浓度 50mM Tris(pH7.5) 50mM NaCl 1mM MgCl 2
      0.05%Nonidet P40
    2. 用NT2缓冲液洗涤珠子。 如果你有4个样品,你需要取100微升的珠子每个Eppendorf管,共4管。 3,600rpm,在4℃下1分钟,除去上清液。 用200μlNT2缓冲液洗涤3次
    3. 向每个管中加入30μg靶抗体(例如来自NBL的HuR1μg/μl)
    4. 每管加入320μlNT2缓冲液。 夜间在4℃下旋转。
    第2天
    1. 在4℃下将管(Ab +珠)离心5000×g 5分钟。 丢弃上清液用新鲜制备的NT2缓冲液1ml洗涤两次。 最后一次洗涤后,暂时保留上清液,放在冰上,直到细胞裂解液准备好。

  2. 哺乳动物细胞裂解
    1. 准备细胞沉淀:
      1. 如果需要,通过吸液或使用细胞刮刀从培养皿中分离细胞。 收集细胞悬浮液在离心管中。
      2. 在4℃下将细胞悬浮液以300×g离心5分钟以沉淀细胞。 小心取出并弃去上清液。
      3. 通过用冰冷的PBS重悬细胞沉淀来洗涤细胞。
      4. 在4℃下将细胞悬浮液以300×g离心5分钟以沉淀细胞。 小心取出并弃去上清液。
      5. 使用步骤3-4再次洗涤细胞。
      6. 通过用冰冷的无核酸酶的PBS重悬细胞沉淀来洗涤细胞。
      7. 在4℃下将细胞悬浮液以300×g离心5分钟以沉淀细胞。 小心取出并弃去上清液。
      8. 通过用冰冷的无核酸酶的PBS重悬细胞沉淀来清洗细胞
      9. 等分细胞悬浮液到每个新的微量离心管。
      10. 在4℃下将细胞悬浮液以300×g离心5分钟以沉淀细胞。 小心取出并弃去上清液。
    2. 裂解细胞沉淀
      1. 在最终以下浓度的DEPEC处理水中制备RSB缓冲液 10mM Tris(pH7.5) 100 mM NaCl
        2.5mM MgCl 2 v/v 然后在每ml RSB缓冲液中加入其他补充剂。
        10μldigitornin/ml缓冲液(Digitornin新鲜溶于乙醇4mg/ml),3μlRNA酶抑制剂/ml缓冲液,40μl蛋白酶抑制剂混合物/ml。
      2. 加入200微升的RSB缓冲液到每个细胞沉淀。 在冰上2分钟。 离心机4,400rpm 4℃,8分钟。
      3. 为每个管(Ab +珠)制作753μl主混合物。 对于4管,你可以做4.5倍以上 700μlNT2缓冲液
        10μl0.1M DTT
        10μlRNA酶抑制剂
        33μl0.5M EDTA(pH8)
      4. 然后将细胞裂解物的每个上清液加入管(Ab +珠)。 在4℃下旋转2小时
      5. 从混合物中取20μl的蛋白质印迹。 离心管在5,000×g 4℃2分钟。
      6. 弃去上清液。 用1ml NT2缓冲液在5,000xg 4℃2分钟洗涤沉淀两次。
      7. 进一步处理混合物以获得mRNA。 使DNAase主混合 现在我们有4个管,所以做4.5倍主混合 100μlNT2缓冲液×4.5 =450μl
        5μlDNA酶(2U /μl)×4.5 =22.5μl
        每管加入105μlDNAase主混合物,37℃,10分钟孵育
      8. 然后在4℃下加入1ml NT2缓冲液离心机5000×g 5分钟。 除去上清液。
      9. 添加蛋白酶K(20毫克/微升)主混合103微升到每个管。 55℃,15分钟加混合 主混合物:2.5μl蛋白酶K x 4.5 =11.25μl
        0.5μl20%SDS×4.5 =2.25μl
        100μlNT2缓冲液×4.5 =450μl
      10. 离心机5,000×g <4℃5分钟。
      11. 收集100μl上清液到新的Eppendorf管。 加入200μl的NT2缓冲液到老管,离心5000×g 4℃,再次5分钟。 收集200微升的上清液,并集中到新的管,这变成300微升的上清
      12. 向上清液中加入等体积的酸性苯酚/氯仿 顶点1分钟,然后离心1分钟RT,13k rpm
      13. 收集约250μl水相(上相)
        加入:25μl3M NaoAc(pH 5.5)
        625μl100%乙醇
        3微升糖原蓝
        充分混合,-20℃或以下放置20分钟(如有必要,过夜)
      14. 在4℃下以12,000×g离心管10分钟,然后小心地抽吸上清液。
      15. 在4℃下用1ml 70%乙醇冲洗,13,000×g 2分钟。 弃去上清液。 倒置管,空气干燥5分钟。 沉淀是mRNAs

食谱

  1. 裂解缓冲液
    在使用前,向裂解缓冲液中加入适当浓度的蛋白酶抑制剂,RNA酶抑制剂和DTT。 含有这些试剂的裂解缓冲液在以下方案中被描述为裂解缓冲液(+)。 用于RIP-测定的每种试剂的最佳浓度如下所示 在DEPEC处理水中制备RSB缓冲液,最终浓度如下:
    10mM Tris(pH7.5) 100 mM NaCl
    2.5mM MgCl 2 v/v 然后在每ml RSB缓冲液中加入其他补充剂。
    10μldigitornin/ml缓冲液(digitornin溶于乙醇4mg/ml新鲜),3μlRNA酶抑制剂/ml缓冲液,40μl蛋白酶抑制剂混合物/ml。
  2. 洗涤缓冲液
    恰好在使用前向洗涤缓冲液中加入最终1.5mM DTT。 含有DTT的洗涤缓冲液在以下方案中称为洗涤缓冲液(+) 在DEPEC处理水中制备洗涤缓冲液,最终浓度如下:
    50mM Tris(pH7.5) 50mM NaCl 1mM MgCl 2
    0.05%Nonidet P40
  3. 注意:附加缓冲液准备
    在一些情况下,裂解缓冲液(+)和洗涤缓冲液(+)都可能需要加入适当体积的高盐溶液(在这些情况下,向每ml裂解缓冲液中加入30μl高盐溶液, 缓冲区)。

致谢

该协议改编自NBL RIP-测定试剂盒(参见参考文献1)。

参考文献

  1. 来自NBL RIP-测定试剂盒(NBL,目录号:RN1001)的方案。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, F. (2012). RNP-IP (Modified Method)-Getting Majority of RNA from RNA Binding Protein in Cytoplasm. Bio-protocol 2(13): e219. DOI: 10.21769/BioProtoc.219.
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