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miRNA Tagging and Affinity-purification (miRAP)
miRNA 标记和亲和纯化(MiRAP)

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Abstract

MicroRNAs(miRNAs) are a group of endogenously expressed 20~23 nt small noncoding RNAs, which can directly regulate mRNA stability or translation in a sequence specific manner by incomplete base pairing at the 3’UTR of target mRNA, or indirectly affect transcriptional network by regulating transcription factors. As key regulators of gene expression, miRNAs are involved in the control of diverse developmental and physiological processes, including embryogenesis, differentiation, developmental timing, organogenesis, growth control, and programmed cell death. Aberrant miRNA expression profiles have been observed in many pathological conditions, including cancers, psychiatric diseases, virus infection, etc. However, the underlying mechanisms have been difficult to study in part due to the cellular heterogeneity of complex tissue.
To systematically analyze miRNA expression in complex tissue, we present here a novel miRNA tagging and Affinity Purification method, miRAP, which can be applied to genetically defined cell types in any complex tissues in mice. This method is based on the fact that mature miRNAs are incorporated into RNA-induced silencing complex (RISC), in which the Argonaute protein AGO2 directly binds miRNAs and their mRNA targets. We demonstrate that epitope tagging of AGO2 protein allows direct purification of miRNAs from tissue homogenates using antibodies against the engineered molecular tag. We further established a Cre-loxP binary expression system to deliver epitope-tagged AGO2 (tAGO2) to genetically defined cell types.

Keywords: MicroRNA(microRNA), Agonaute(agonaute), Expression profile(表达谱), Neuron(神经元), Immunoprecipitation(免疫共沉淀)

Materials and Reagents

  1. Mouse-anti-c-Myc (Santa Cruz, catalog number: sc-40 )
  2. Mouse-anti-Ago2 (Clone 2E12-1C9) (Abnova, catalog number: H00027161-M01 )
  3. Rabbit-anti-GFP (Rockland, catalog number: 600-401-215 ) or Chicken-anti-GFP (Rockland, catalog number: 600-901-215 )
  4. Mouse IgG1 negative control (clone Ci4) (EMD Millipore)
    Note: The above antibodies have been tested by the author and may be substituted with the antibodies desired by users.
  5. Complete proteinase inhibitors (EDTA-free) (Roche Diagnostics)
  6. Protein G Dynabeads (Life Technologies, Invitrogen™)
  7. RNasin(Life Technologies, Ambion®)
  8. Proteinase K (Roche Diagnostics)
  9. Acid phenochloroform (Life Technologies, Ambion®)
  10. Chloroform (Life Technologies, Ambion®)
  11. 3 M Sodium Acetate (pH 5.5) (Life Technologies, Ambion®)
  12. Glycoblue (Life Technologies, Ambion®)
  13. RNAzap (Life Technologies, Ambion®)
  14. DEPC treated water or RNase free water (Life Technologies, Ambion®)
  15. HEPES (pH 7.4)
  16. KCl
  17. MaCl2
  18. NP-40
  19. DTT
  20. EDTA
  21. SDS
  22. Lysis buffer (see Recipes)
  23. Low salt NT2 buffer (see Recipes)
  24. High salt NT2 buffer (see Recipes)
  25. 0.5% NP-40 (see Recipes)
  26. Proteinase K buffer (see Recipes)

Equipment

  1. Glass douncer
  2. Mortar and pestle
  3. Ice bucket
  4. Rotator
  5. 4 °C centrifuge
  6. Standard western blot set up

Procedure

  1. Activate and validate tAGO2 expression in the cell of interest
    1. Set up appropriate Cre driver line breeding with LSL-tAgo2 reporter line (JAX stock number: 017626) to express tAgo2 in the cell of interest.
    2. Verify tAgo2 expression in the cell of interest by co-immunostaining of GFP tag within tAgo2 and markers identifying that cell type in tissue sections. Recommended dilution of GFP antibody is 1:800~1:1,000.
    3. Euthanize the mouse and dissect out tissue of interest on ice as soon as possible.
    4. Flash freeze tissue in liquid nitrogen (pause point: Tissue block can be stored in liquid nitrogen for at least half year).

  2. miRAP sample preparation
    Note: It is important to work in an RNase free environment from this part on. Gloves should be worn at all time. Bench top should be wiped with RNAzap. Glassware should be cleaned with RNAzap and rinsed with DEPC-treated water or RNase free water. RNase free pipette tips and tubes should be used when handling the samples. All reagents should be prepared in DEPC-treated water or RNase free water.
    1. Cool down mortar and pestle in a liquid nitrogen containing ice bucket.
    2. Pour appropriate amount of liquid nitrogen into the mortar (enough to immerse tissue block, but not too much that the liquid nitrogen will spill out), ground tissue into fine powder.
    3. Transfer the tissue powder along with liquid nitrogen into a 50 ml falcon tube, loosely cap the tube, let it sit in room temperature for a few minutes until the liquid nitrogen completely evaporate.
    4. Add 10 volume of lysis buffer, resuspend tissue powder quickly and transfer into pre-cooled glass douncer.
    5. Homogenized tissue suspension using glass douncer by douncing 50-100 times. Certain tissue may take longer to lyse. Adjust the number of douncing according to your application.
    6. Transfer tissue homogenates into 1.5 ml or 2.0 ml eppendorf tubes, centrifuge at 13,000 x g for 30 min, 4 °C to pellet cell debris and unsolubilized material.
    7. Transfer supernatant to a new tube. This will be the sample to use for miRAP.

  3. miRNA affinity purification
    1. Prepare antibody conjugated protein G Dynabeads according to manufacturer's instruction. For cell type specific miRAP, use Myc antibody; for whole tissue control, use Ago2 antibody; for negative control, use mouse IgG1. The amount of beads to be used per sample and the antibody to beads ratio should be empirically determined. As a starting point, use 10 μg Myc antibody, 2.5 μg Ago2 antibody, and 10 μg IgG1 per 50 μl beads.
    2. Add antibody conjugated beads to IP sample and incubate the mixture in 4 °C with end-over-end rotation for 4 h. The amount of beads to use per sample should be determined empirically. Western blot can be used to check the efficiency of IP. If there is still residue Ago2 or tAgo2 present in the supernatant after IP, use more beads.
    3. Wash beads twice with low salt NT2 buffer and twice with high salt NT2 buffer, 15 min each, and treat the beads with 0.6 mg ml-1 proteinase K for 20 min at 55 °C.
    4. Add equal volume of acid phenochloroform to the sample, mix well by vortexing, and spin at maximum speed in 4 °C for 15 min; transfer the upper phase to a new tube without disturbing the interphase. Repeat this extraction procedure twice with chloroform instead of phenochloroform to get rid of residue phenol. Add at least 3 volume of 100% ethanol, 1/10 volume of sodium acetate (pH 5.2), and 1 μl of glycoblue to the transferred upper phase, mix well, and precipitate RNA overnight at -80 °C.
    5. Wash RNA pellet once with 75% ethanol and dissolve it in water for further application. (Pause point: RNA pellet can be stored in 75%-100% ethanol in -80 °C for years. RNA solution can be stored in -80 °C, but multiple freeze-thaw cycles should be avoided.)

  4. Downstream applicationmiRNA purified from miRAP has high quality and can be directly used for deep sequencing, miRNA Taqman PCR, miRNA microarrary, Northern blot (which requires a high input and may not be possible for rare cell types or low expression miRNAs), etc., following standard protocol.
    Note: It is recommended to use Taqman PCR for a quick and sensitive examination of candidate miRNAs before you proceed to sequencing or microarray.

Recipes

  1. Lysis buffer
    10 mM HEPES (pH 7.4)
    100 mM KCl
    5 mM MaCl2
    0.5% NP-40
    1 mM DTT
    100 U ml-1 RNasin
    Roche Complete proteinase inhibitors EDTA-free (1 tab/10 ml).
  2. Low salt NT2 buffer
    50 mM Tris-HCl (pH 7.5)
    150 mM NaCl
    1 mM MgCl2
    0.5% NP-40
    1 mM DTT
    100 U ml-1 RNasin
  3. High salt NT2 buffer
    50 mM Tris-HCl (pH 7.5)
    600 mM NaCl
    1 mM MgCl2
  4. 0.5% NP-40
    1 mM DTT
    100 U ml-1 RNasin
  5. Proteinase K buffer
    100 Mm Tris-HCl (pH 7.5)
    50 mM NaCl
    10 mM EDTA
    0.5% SDS
    10 mg ml-1 proteinase K

Acknowledgments

This protocol is based on the published paper He et al. (2012).

References

  1. Bartel, D. P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2): 281-297.
  2. He, L. and Hannon, G. J. (2004). MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5(7): 522-531.
  3. He, M., Liu, Y., Wang, X., Zhang, M. Q., Hannon, G. J. and Huang, Z. J. (2012). Cell-type-based analysis of microRNA profiles in the mouse brain. Neuron 73(1): 35-48.

简介

微小RNA(miRNA)是一组内源性表达的20〜23nt的小非编码RNA,其可以通过在靶mRNA的3'UTR处的不完全碱基配对以序列特异性方式直接调节mRNA稳定性或翻译,或间接影响转录网络调节转录因子。作为基因表达的关键调节剂,miRNA参与多种发育和生理过程的控制,包括胚胎发生,分化,发育时间,器官发生,生长控制和程序性细胞死亡。在许多病理状况,包括癌症,精神疾病,病毒感染等中已观察到异常的miRNA表达谱。然而,部分由于复杂组织的细胞异质性,潜在的机制难以研究。
为了系统地分析复杂组织中的miRNA表达,我们在这里提出了一种新的miRNA标签和亲和纯化方法,miRAP,其可以应用于小鼠中任何复杂组织中的遗传定义的细胞类型。这种方法基于这样的事实,即成熟的miRNA掺入RNA诱导的沉默复合物(RISC),其中Argonaute蛋白AGO2直接结合miRNA和它们的mRNA靶。我们证明表位标签的AGO2蛋白允许直接从组织匀浆使用针对工程分子标签的抗体的miRNA纯化。我们进一步建立Cre-loxP二元表达系统,以将表位标记的AGO2(tAGO2)递送到遗传定义的细胞类型。

关键字:microRNA, agonaute, 表达谱, 神经元, 免疫共沉淀

材料和试剂

  1. 小鼠抗c-Myc(Santa Cruz,目录号:sc-40)
  2. 小鼠抗Ago2(克隆2E12-1C9)(Abnova,目录号:H00027161-M01)
  3. 兔抗GFP(Rockland,目录号:600-401-215)或鸡抗GFP(Rockland,目录号:600-901-215)
  4. 小鼠IgG1阴性对照(克隆C14)(EMD Millipore)
    注意:上述抗体已经被作者测试,可能被用户需要的抗体替代。
  5. 完全蛋白酶抑制剂(不含EDTA)(Roche Diagnostics)
  6. 蛋白G Dynabeads(Life Technologies,Invitrogen TM)
  7. RNasin(Life Technologies,Ambion
  8. 蛋白酶K(Roche Diagnostics)
  9. 酸性酚氯仿(Life Technologies,Ambion )
  10. Chloroform(Life Technologies,Ambion
  11. 3 M乙酸钠(pH 5.5)(Life Technologies,Ambion )
  12. Glycoblue(Life Technologies,Ambion )
  13. RNAzap(Life Technologies,Ambion )
  14. DEPC处理的水或不含RNase的水(Life Technologies,Ambion )
  15. HEPES(pH 7.4)
  16. KCl
  17. MaCl 2
  18. NP-40
  19. DTT
  20. EDTA
  21. SDS
  22. 裂解缓冲液(见配方)
  23. 低盐NT2缓冲液(参见配方)
  24. 高盐NT2缓冲液(见配方)
  25. 0.5%NP-40(见配方)
  26. 蛋白酶K缓冲液(见配方)

设备

  1. 玻璃douncer
  2. 砂浆和杵
  3. 冰桶
  4. 旋转器
  5. 4℃离心机
  6. 标准蛋白质印迹设置

程序

  1. 激活和验证感兴趣的细胞中的tAGO2表达
    1. 用LSL-tAgo2报告系(JAX库存号:017626)设置适当的Cre驱动系育种以在目的细胞中表达tAgo2。
    2. 验证感兴趣的细胞中的tAgo2表达通过GFP标签在tAgo2内的共免疫染色和标记组织切片中的细胞类型的标记。 GFP抗体的推荐稀释度为1:800〜1:1000。
    3. 安乐死鼠标,并尽快在冰上解剖感兴趣的组织
    4. 在液氮中闪烁冻结组织(暂停点:组织块可以储存在液氮中至少半年)。

  2. miRAP样品准备
    注意:从本部分开始,在不含RNase的环境中工作很重要。 手套应始终佩戴。 台面应该用RNAzap擦拭。 玻璃器皿应使用RNAzap清洁,并用DEPC处理的水或冲洗 不含RNase的水。处理样品时应使用不含RNase的移液管吸头和管。所有试剂应在DEPC处理的水或不含RNase的水中制备。
    1. 在含有液氮的冰桶中冷却研钵和杵。
    2. 将适量的液氮倒入砂浆中(足够浸入组织块,但不要过多,以致液氮会溢出),将组织磨成细粉。
    3. 将组织粉末与液氮一起转移到50ml的falcon管中,松松地盖上管,让其在室温下放置几分钟直到液氮完全蒸发。
    4. 加入10倍体积的裂解缓冲液,迅速重悬浮组织粉,转移到预冷的玻璃管中
    5. 均质组织悬浮液使用玻璃douncer通过douncing 50-100次。某些组织可能需要更长的时间来裂解。根据您的应用调整滴定次数。
    6. 将组织匀浆转移到1.5ml或2.0ml eppendorf管中,在13,000×g离心30分钟,4℃以沉淀细胞碎片和未溶解的材料。
    7. 将上清转移到新管中。这将是用于miRAP的样品。

  3. miRNA亲和纯化
    1. 根据制造商的说明制备抗体偶联的蛋白G Dynabeads。对于细胞类型特异性miRAP,使用Myc抗体;对于全组织控制,使用Ago2抗体;对于阴性对照,使用小鼠IgG1。每个样品使用的珠子的量和抗体珠子的比率应该根据经验确定。作为起点,使用10μgMyc抗体,2.5μgAgo2抗体和10μgIgG1 /50μl珠子。
    2. 向IP样品中加入抗体偶联的珠子,并在4℃下,将混合物在末端旋转下孵育4小时。每个样品使用的珠的量应该根据经验确定。 Western印迹可用于检查IP的效率。如果IP后仍有上清液中残留的Ago2或tAgo2,则使用更多的珠子
    3. 用低盐NT2缓冲液洗涤珠两次,用高盐NT2缓冲液洗涤两次,每次15分钟,并在55℃下用0.6mg/ml蛋白酶K处理珠20分钟。 >
    4. 向样品中加入等体积的酸性酚氯仿,通过涡旋混合均匀,并在4℃以最大速度旋转15分钟;将上层相转移到新管而不干扰相间。用氯仿代替苯酚氯仿重复该萃取程序两次,以除去残余物苯酚。向转移的上层相中加入至少3体积的100%乙醇,1/10体积乙酸钠(pH 5.2)和1μl糖醇,混匀,在-80℃下沉淀RNA过夜。
    5. 用75%乙醇洗涤RNA沉淀一次,并将其溶解在水中用于进一步的应用。 (暂停点:RNA沉淀可以在75°C -100%乙醇中储存多年,RNA溶液可以储存在-80°C,但应避免多次冻融循环。)

  4. 从miRAP纯化的下游应用miRNA具有高质量,并且可以直接用于深度测序,miRNA Taqman PCR,miRNA微阵列,Northern印迹(其需要高输入并且可能不可能用于稀有细胞类型或低表达miRNA) ,遵循标准协议。
    注意:建议在进行测序或微阵列之前,使用Taqman PCR快速和灵敏地检测候选miRNA。

食谱

  1. 裂解缓冲液
    10mM HEPES(pH7.4) 100 mM KCl
    5mM MaCl 2
    0.5%NP-40
    1 mM DTT
    100 U ml -1 RNasin
    Roche完全蛋白酶抑制剂无EDTA(1片/10ml)
  2. 低盐NT2缓冲液
    50mM Tris-HCl(pH7.5) 150mM NaCl 1mM MgCl 2
    0.5%NP-40
    1 mM DTT
    100 U ml -1 RNasin
  3. 高盐NT2缓冲液
    50mM Tris-HCl(pH7.5) 600 mM NaCl
    1mM MgCl 2
  4. 0.5%NP-40
    1 mM DTT
    100 U ml -1 RNasin
  5. 蛋白酶K缓冲液
    100 Mm Tris-HCl(pH 7.5)
    50mM NaCl 10 mM EDTA
    0.5%SDS
    10mg/ml蛋白酶K

致谢

该协议基于发表的论文He (2012)。

参考文献

  1. Bartel,D.P。(2004)。 MicroRNA:基因组学,生物发生,机制和功能。 116(2):281-297。
  2. He,L。和Hannon,G.J。(2004)。 MicroRNA:在基因调控中发挥重要作用的小RNA。 Nat Rev Genet 5(7):522-531。
  3. He,M.,Liu,Y.,Wang,X.,Zhang,M. Q.,Hannon,G.J.and Huang,Z.J.(2012)。 基于细胞类型的小鼠脑中microRNA谱的分析。 Neuron 73(1):35-48
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引用:He, M. (2012). miRNA Tagging and Affinity-purification (miRAP). Bio-protocol 2(19): e265. DOI: 10.21769/BioProtoc.265.
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Justin Slawson
Columbia University/Biogen Idec
Great paper, this is really a useful technique! 2 quick questions: 1) For step 3d, do you remove the beads before adding the acid phenolchloroform? Or do you add it directly to the bead/RNA mixture? and 2) Have you ever tried any commercially available RNA extraction kits instead of the phenolchloroform step? It looks to me like the miRNeasy kit from Qiagen follows a pretty similar extraction protocol as you, except that it uses spin columns. Just wondering if there are any reasons to avoid the Qiagen kits for this protocol. Thank you so much, and thanks for putting together this great protocol!

Justin
1/16/2014 8:37:21 PM Reply