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Cross-linked RNA Immunoprecipitation
RNA免疫共沉淀   

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Abstract

This method is for the immunoprecipitation of Flag-Tagged RNA binding proteins from mammalian cell lines and isolation of the bound RNAs for analysis by quantitative real-time PCR. The RNA binding protein of interest should be tagged with the M2 Flag-tag and expressed in the mammalian cell line of interest (Knuckles et al., 2012). However, specific antibodies for the protein of interest can be used in conjunction with Sepharose G-beads.

Materials and Reagents

  1. Anti-Flag M2 Affinity gel (Sigma Aldrich, catalog number: A2220 )
  2. RNase inhibitor (Life Technologies, InvitrogenTM, catalog number: N8080119 or Bioline, catalog number: BIO-65028 )
  3. Complete Protease Inhibitor Cocktail Tablets (F. Hoffmann-La Roche, catalog number: 05 892 970 001 )
  4. RnaseZap (Life Technologies, Ambion®, catalog number: AM9780 )
  5. Trizol reagent (Life Technologies, InvitrogenTM, catalog number: 15596-026 )
  6. General chemicals (Sigma Aldrich)
  7. DNase I recombinant, RNase-free inc. buffer (F. Hoffmann-La Roche, catalog number: 04716728001 )
  8. BioScriptTM (Bioline, catalog number: BIO-27036 )
  9. Formaldehyde 
  10. Fetal calf serum
  11. Glycine
  12. SDS
  13. HEPES
  14. TritonX-100
  15. EDTA
  16. DTT
  17. NaCl
  18. IP lysis buffer (see Recipes)
  19. RIP buffer (see Recipes)

Equipment

  1. Sonicator (the model is not critical but preferably a devise with a probe ≤ 5 mm in diameter)
  2. Tube Rotator
  3. Shaker
  4. Centrifuge
  5. PCR machine
  6. Heating block
  7. 15 ml conical tubes

Procedure

  1. Preparation of beads and cells
    1. Blocking of the beads:
      Wash 40 μl Anti-Flag M2 Affinity Gel twice with 900 μl of pure H2O (cold); add 400 μl IP lysis buffer +1% BSA; incubate at 4 °C (on a rotating wheel) overnight.
      Alternatively use 30 μl SepharoseG-Beads + specific antibody. The amount of antibody and beads to be used in the precoupling will need to be determined in preliminary experiments. Typically using 10 μg of Ig for coupling is a reasonable starting point.
    2. Use 2 x 10 cm2 tissue culture plate per condition (this might vary depending on the cell type and the level of expression of the protein of interest). Do not process more than 10 plates at a time.
    3. Transfect cells and incubate them for 48 h to express the tagged protein

  2. Harvesting the cells
    1. Remove medium, wash the cells once with phosphate buffered saline and add 1 ml 0.25% Trypsin (pre-heated to 37 °C). Incubate at 37 °C until the cells start to detach.
    2. Add 5 ml DMEM including 10% fetal calf serum (pre-warmed to 37 °C) to inhibit the trypsin.
    3. Remove the cells from the plate by pipetting.
    4. Transfer the cells to a 15 ml conical tube and incubate on ice for 5 min.
    5. Harvest cells 2 min at 100 x g at room temperature, decant the supernatant and resuspend the cell pellet in 5 ml ice-cold phosphate buffered saline. Keep a 250 μl aliquot for Western blot analysis to be used as a transfection control.
    6. Add 143 μl 37% formaldehyde (over a period of approximately 10 sec, treat all of the samples in the same way).
    7. Place the 15 ml conical tubes on a rocking plate and shake for 10 min at room temperature.
    8. Add 685 μl 2 M glycine (over a period of approximately 10 sec, treat all of the samples in the same way) to block the formaldehyde.
    9. Place the 15 ml conical tubes on a rocking plate and shake for 5 min at room temperature.
    10. Harvest cells by centrifugation for 2 min at 100 x g at room temperature.
    11. Decant the supernatant and transfer the cell pellet to ice.
    12. Wash the cells twice with 5 ml ice-cold phosphate buffered saline and harvest the cells 2 min at 100 x g at 4 °C.
    13. Remove the supernatant from the cells after the last wash and add 1 ml of IP lysis buffer + 20 μl 0.1 M phenylmethylsulfonyl fluoride + 20 μl complete protease inhibitor (50x) + 5 μl RNase inhibitor (40 U/μl) to each sample.
    14. Sonicate the cells, keeping them on ice at all times, 10x (10 sec on, 10 sec off, Amplitude 15 μm) until the lysate is clear. Clean probe sonicator with RNaseZap between the samples to avoid contamination with RNases.
    15. Transfer the cells to a 1.5 ml snap-cap tube and keep on ice.
    16. Centrifuge the lysates for 3 min at 14,000 x g at room temperature and keep 50 μl of the supernatant as an INPUT control (used to standardize the qPCR analysis).

  3. Immunoprecipitation
    1. Add the rest of the lysate to the blocked beads and incubate overnight at 4 °C on a rotating wheel.
    2. Wash the beads 5 times with 900 μl of IP lysis buffer collecting the beads by centrifugation for 1 min at 400 x g at room temperature.
    3. Remove all of the supernatant after the last wash and add 100 μl of RIP buffer + 1 μl RNase inhibitor.
      Add 50 μl of RIP buffer to the INPUT control (step 18).
    4. Incubate the samples and INPUT controls for 1 h at 70 °C to reverse the cross-link.
    5. Centrifuge for 1 min at 400 x g at room temperature to sediment the beads and collect 100 μl of supernatant.
    6. Extract the RNA with Trizol reagent (proceed according to the manufacturer’s instruction (Invitrogen)). Optional: Add 1 μl of Glycogen blue before precipitation of the RNA with Isopropanol to make the pellet visible. Precipitation can also be performed at -20 °C for 1 h to increase the amount of precipitated RNA.
    7. DNAse treatment of the RNA to avoid contamination with genomic DNA.
      Dilute the RNA pellet directly in DNase mastermix (per sample: 16 μl DEPC treated H2O, 2 μl DNaase buffer, 2 μl DNAse) in order to use all RNA in the following RT reaction and proceed after the manufacturer’s instruction (Roche).

  4. Reverse transcription of the RNA into cDNA
    1. BioScriptTM (Bioline) kit works well but other reverse transcriptase kits may also be used.
      9 μl of the DNAse treated RNA was used in a reverse transcriptase-containing and a reverse transcriptase-minus (negative control) reaction for each sample. cDNA was primed with random hexamer primers and the reaction performed according to the manufacturer’s instructions.
    2. Dilute the cDNA 1:4 and continue with quantitative real-time PCR analysis of target gene to identify specific changes in target mRNA level. Alternatively the cDNA can be used to generate a library.

Recipes

  1. IP lysis buffer
    50 mM Hepes (pH 7.5)
    0.4 M NaCl
    1 mM EDTA
    1 mM DTT
    0.5% TritonX-100
    10% Glycerol
  2. RIP buffer
    50 mM Hepes (pH 7.5)
    0.1 M NaCl
    5 mM EDTA
    10 mM DTT
    0.5% TritonX-100
    10% Glycerol
    1% SDS
    All solutions should be prepared RNAse free with Diethylpyrocarbonate (DEPC)-treated water and autoclaved before use.

Acknowledgments

This protocol was previously used in Knuckles et al. (2012).

References

  1. Knuckles, P., M. A. Vogt, S. Lugert, M. Milo, M. M. Chong, G. M. Hautbergue, S. A. Wilson, D. R. Littman and V. Taylor (2012). Drosha regulates neurogenesis by controlling neurogenin 2 expression independent of microRNAs. Nat Neurosci 15(7): 962-969.

简介

该方法用于来自哺乳动物细胞系的Flag-标记的RNA结合蛋白的免疫沉淀和分离结合的RNA,用于通过定量实时PCR分析。 感兴趣的RNA结合蛋白应该用M2 Flag标签标记并在感兴趣的哺乳动物细胞系中表达(Knuckles等人,2012)。 然而,感兴趣的蛋白质的特异性抗体可以与Sepharose G-珠结合使用。

材料和试剂

  1. 抗Flag M2亲和凝胶(Sigma Aldrich,目录号:A2220)
  2. RNase抑制剂(Life Technologies,Invitrogen TM,目录号:N8080119或Bioline,目录号:BIO-65028)
  3. 完全蛋白酶抑制剂混合片(F.Hoffmann-La Roche,目录号:05 892 970 001)
  4. RnaseZap(Life Technologies,Ambion ,目录号:AM9780)
  5. Trizol试剂(Life Technologies,Invitrogen TM,目录号:15596-026)
  6. 一般化学品(Sigma Aldrich)
  7. DNase I重组,RNase-free inc。 缓冲液(F.Hoffmann-La Roche,目录号:04716728001)
  8. BioScript TM(Bioline,目录号:BIO-27036)
  9. 甲醛
  10. 胎牛血清
  11. 甘氨酸
  12. SDS
  13. HEPES
  14. TritonX-100
  15. EDTA
  16. DTT
  17. NaCl
  18. IP裂解缓冲液(参见配方)
  19. RIP缓冲区(参见配方)

设备

  1. 超声波仪(该型号并不重要,但最好是一个直径≤5 mm的探头)
  2. 管式旋转器
  3. 振动器
  4. 离心机
  5. PCR机
  6. 加热块
  7. 15 ml锥形管

程序

  1. 珠和细胞的制备
    1. 珠子的阻塞:
      用900μl纯H 2 O(冷)洗涤40μl抗Flag M2亲和凝胶两次; 加入400μlIP裂解缓冲液+ 1%BSA; 在4℃(旋转轮上)温育过夜 或者使用30μlSepharose G-Beads +特异性抗体。 在预偶联中使用的抗体和珠子的量需要在初步实验中确定。 通常使用10μg的Ig进行偶联是一个合理的起点
    2. 每个条件使用2×10cm 2组织培养板(这可能根据细胞类型和感兴趣的蛋白质的表达水平而变化)。 一次不要处理超过10块板。
    3. 转染细胞并孵育48小时以表达标记的蛋白质

  2. 收获细胞
    1. 除去培养基,用磷酸盐缓冲盐水洗涤细胞一次,并加入1ml 0.25%胰蛋白酶(预热至37℃)。 在37℃孵育,直到细胞开始分离。
    2. 加入5ml含有10%胎牛血清(预热至37℃)的DMEM以抑制胰蛋白酶。
    3. 通过移液除去板中的细胞。
    4. 转移细胞到15毫升锥形管,在冰上孵育5分钟
    5. 在室温下以100×g收获细胞2分钟,倾析上清液并将细胞沉淀重悬于5ml冰冷的磷酸盐缓冲盐水中。 保留250μl等分试样用于Western印迹分析以用作转染对照
    6. 加入143μl37%甲醛(在约10秒的时间内,以相同的方式处理所有样品)
    7. 将15毫升锥形管放在摇摆板上,在室温下摇动10分钟
    8. 加入685μl2 M甘氨酸(在约10秒的时间内,以相同的方式处理所有样品)以阻断甲醛。
    9. 将15毫升锥形管放在摇摆板上,在室温下摇动5分钟
    10. 通过在室温下以100×g离心2分钟收获细胞
    11. 倾析上清液并将细胞沉淀转移到冰上
    12. 用5ml冰冷的磷酸盐缓冲盐水洗涤细胞两次,并在4℃以100×g收集细胞2分钟。
    13. 在最后一次洗涤后从细胞中除去上清液,并向每个样品加入1ml IP裂解缓冲液+20μl0.1M苯甲基磺酰氟+20μl完全蛋白酶抑制剂(50x)+5μlRNA酶抑制剂(40U /μl) br />
    14. 超声细胞,保持他们在冰的所有时间,10x(10秒开,10秒关闭,振幅15微米),直到裂解物清除。 用样品之间的RNaseZap清洁探针超声波仪,以避免RNase污染
    15. 将细胞转移到1.5 ml snap-cap管,并保持在冰上
    16. 在室温下以14,000×g离心裂解物3分钟,并保持50μl上清液作为INPUT对照(用于标准化qPCR分析)。

  3. 免疫沉淀
    1. 将剩余的裂解物加入封闭的珠中,并在4℃下在旋转轮上孵育过夜
    2. 用900μlIP裂解缓冲液洗涤珠子5次,通过在室温下以400×g离心1分钟收集珠子。
    3. 在最后一次洗涤后取出所有上清液,加入100μlRIP缓冲液+ 1μlRNA酶抑制剂 向INPUT控制中添加50μlRIP缓冲液(步骤18)。
    4. 将样品和INPUT对照在70°C孵育1小时,以逆转交联
    5. 在室温下以400×g离心1分钟以沉淀珠子并收集100μl上清液。
    6. 可选:在用异丙醇沉淀RNA之前,添加1μl糖原蓝,以使沉淀可见。使用Trizol试剂提取RNA(根据制造商的说明书(Invitrogen))。沉淀也可以在-20℃进行1小时,以增加沉淀的RNA的量
    7. DNA酶处理RNA以避免污染基因组DNA。
      在DNase mastermix(每个样品:16μlDEPC处理的H 2 O,2μlDNaase缓冲液,2μlDNAse)中直接稀释RNA沉淀,以便在以下RT反应中使用所有RNA,并在制造商的说明(Roche)
  4. 将RNA逆转录成cDNA
    1. BioScript?sup?TM(Bioline)试剂盒效果很好,但也可以使用其他逆转录酶试剂盒。
      将9μlDNAse处理的RNA用于每个样品的含逆转录酶和逆转录酶 - 阴性(阴性对照)反应中。用随机六聚体引物引发cDNA,并根据制造商的说明书进行反应
    2. 稀释cDNA 1:4,继续对靶基因进行定量实时PCR分析,以鉴定靶mRNA水平的特异性变化。或者,cDNA可用于产生文库

食谱

  1. IP裂解缓冲液
    50mM Hepes(pH7.5) 0.4 M NaCl
    1mM EDTA
    1 mM DTT
    0.5%TritonX-100 10%甘油
  2. RIP缓冲区
    50mM Hepes(pH7.5) 0.1 M NaCl
    5 mM EDTA
    10 mM DTT
    0.5%TritonX-100 10%甘油
    1%SDS
    所有溶液应制备无RNA酶的焦磷酸二乙酯(DEPC)处理的水,并在使用前进行高压灭菌。

致谢

此协议以前在Knuckles等人使用。(2012)。

参考文献

  1. Knuckles,P.,M. A. Vogt,S. Lugert,M. Milo,M. M. Chong,G. M. Hautbergue,S. A. Wilson,D. R. Littman and V. Taylor(2012)。 Drosha通过控制独立于microRNA的神经元2表达来调节神经发生。 Nat Neurosci 15(7):962-969。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Vogt, M. and Taylor, V. (2013). Cross-linked RNA Immunoprecipitation. Bio-protocol 3(5): e398. DOI: 10.21769/BioProtoc.398.
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