mRNA Stability Assay Using Transcription Inhibition by Actinomycin D in Mouse Pluripotent Stem Cells
利用放线菌素D抑制转录检测小鼠多能干细胞中mRNA的稳定性   

引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
eLIFE
May 2018

 

Abstract

Gene expression is regulated through multiple steps at both transcriptional and post-transcriptional levels. The net abundance of mature mRNA species in cells is determined by the balance between transcription and degradation. Thus, the regulation of mRNA stability is a key post-transcriptional event that can greatly affect the net level of mRNAs in cells. The mRNA stability within cells can be measured indirectly by analyzing the mRNA half-life following transcription inhibition, where changes in mRNA levels are assumed to reflect mRNA degradation. Determination of mRNA half-life as a measure of mRNA stability is useful in understanding gene expression changes and underlying mechanisms regulating the level of transcripts at different physiological conditions or developmental stages. The protocol described here presents the analysis of mRNA decay as a measure for determining mRNA stability after transcriptional inhibition with Actinomycin D treatment in control and SRSF3 depleted mouse induced pluripotent stem cells (iPSC).

Keywords: mRNA stability (mRNA稳定性), Actinomycin D (放线菌素 D), mRNA decay (mRNA降解), Transcription inhibitors (转录抑制剂), mRNA half-life (mRNA半衰期)

Background

Determining the stability of mRNA within cells provides an important measure for understanding post-transcriptional gene regulation and the potential role of RNA-protein interactions in the process. Under any specific condition such as following extracellular stimuli or gene knockdown the stability of mRNAs may change due to enhanced degradation or extended half-lives (Shyu et al., 1989). Therefore, to assess mRNA stability, direct measurements of decay rates of endogenous mRNAs have been performed in a number of ways, including kinetic labeling techniques and the use of transcriptional inhibitors (Chen et al., 2008). One of the simplest techniques of measuring mRNA stability is by inhibiting transcription in vivo with transcription inhibitors and measuring the mRNA kinetics.

Actinomycin D is a transcription inhibitor which intercalates into DNA. Actinomycin D forms a very stable complex with DNA, preventing the unwinding of the DNA double-helix, thus inhibiting the DNA-dependent RNA polymerase activity. Actinomycin D is widely used in mRNA stability assays to inhibit the synthesis of new mRNA, allowing the assessment of mRNA decay by measuring mRNA abundance following transcription inhibition (Avendano and Menéndez, 2008). At low concentrations, Actinomycin D inhibits transcription without significantly affecting DNA replication or protein synthesis (Berg et al., 2002). Other transcription inhibitors such as 5,6-dichloro-1β-1-ribofuranosylbenzimidazole (DRB) which interacts directly with the RNA polymerase II have also been successfully used in similar assays (Harrold et al., 1991). Please refer to Bensaude (2011) for more detailed information on different transcription inhibitors that can be used and their specific properties. More advanced techniques such as the use of inducible promoters to control transient transcription have presented advantages over the potential cytotoxic effects of Actinomycin D or other transcription inhibitors in the analysis of mRNA decay (Chen et al., 2008). However, the advantage of Actinomycin D assay is that it does not require the construction and introduction of exogenous genes into cells, and provides a way of measuring stability changes of endogenous mRNAs (Chen et al., 2008).

We have established serine-arginine-rich splicing factor 3 (SRSF3)-RNA interactions as a critical means to co-ordinate gene expression in pluripotent cells (Ratnadiwakara et al., 2018). Further, SRSF3 has been reported to regulate mRNA levels including its own mRNA abundance via alternative splicing coupled to nonsense mediated decay (NMD) (Anko et al., 2012). To determine if SRSF3 affects the production or stability of NMD-sensitive transcript variants in pluripotent cells, we determined mRNA half-lives of SRSF3 target mRNAs in Actinomycin D treated control and SRSF3-depleted pluripotent stem cells (Ratnadiwakara et al., 2018). Several techniques such as Northern blot analysis, in situ hybridization and quantitative PCR can be used to determine the mRNA half-life after transcription inhibition. We used quantitative PCR which allows rapid and sensitive measurement of half-lives of mRNAs across a broad range of expression levels, including low abundant mRNAs. The protocol described here can be used to successfully measure mRNA decay in pluripotent stem cells.

Materials and Reagents

  1. 6-well cell culture plates (Sigma-Aldrich, catalog number: CLS3516)
  2. Serological pipettes 10 ml (Sigma-Aldrich, catalog number: CLS4488)
  3. 15 ml Falcon tubes (Sigma-Aldrich, catalog number: CLS430791)
  4. Sterile filter pipette tips 10 µl, 20 µl, 200 µl, 1,000 µl (Axygen, catalog numbers: TF10LRS, TF20LRS, TF200LRS and TF1000LRS)
  5. Microcentrifuge tubes (Axygen, catalog number: MCT-175-C)
  6. Neptune semi-skirted 96-well plates (VWR, catalog number: 89126-694)
  7. Optically clear adhesive seal sheets (Thermo Fisher Scientific, catalog number: AB-1170)
  8. Knock-Out DMEM (Thermo Fisher Scientific, catalog number: 10829018)
  9. ES cell grade fetal bovine serum (Sigma-Aldrich, catalog number: F9423)
  10. GlutaMAX (Life Technologies, catalog number: 35050-061)
  11. Trypsin 0.25% (Life Technologies, catalog number: 25200-056)
  12. Penicillin-Streptomycin (Life Technologies, catalog number: 15070-063)
  13. Non-Essential Amino Acids (Life Technologies, catalog number: 11140-050)
  14. Beta-Mercapto Ethanol (Life-technologies, catalog number: 21985-023)
  15. Leukaemia inhibitory factor (LIF) (here LIF was produced by the Monash University Protein Production Unit, Australia)
  16. Phosphate-buffered saline (PBS) (Life Technologies, catalog number: 14190-250)
  17. Cells in culture (here mouse induced pluripotent stem cells)
  18. (z)-4-Hydroxytamoxifen (Sigma-Aldrich, catalog number: H7904-5MG)
  19. Cell culture grade Actinomycin D (Sigma-Aldrich, catalog number: A9415) 
  20. Cell culture grade Dimethyl Sulfoxide (DMSO) (AppliChem, catalog number: A3672,0100) 
  21. TRI Reagent (Sigma-Aldrich, catalog number: T9424)
  22. Chloroform (Sigma-Aldrich, catalog number: 288306)
  23. Isopropanol (Sigma-Aldrich, catalog number: 278475)
  24. RNA grade Glycogen (Thermo Fisher Scientific, catalog number: R0551)
  25. Ethanol (any molecular grade)
  26. RNase-free water (Invitrogen, catalog number: 10977-015) 
  27. RQ1 DNase kit (Promega, catalog number: M6101)
  28. SuperScript III Reverse transcription kit (Thermo Fisher Scientific, catalog number: 18080044)
  29. RNaseOUT (Thermo Fisher Scientific, catalog number: 10777019)
  30. Random hexamer primer mix (Bioline, catalog number: BIIO38028)
  31. OligodT18 (IDT)
  32. SYBR green master mix, here Luminaries HiGreen qPCR Master Mix, Low ROX (Thermo Fisher Scientific, catalog number: K0974)
  33. qPCR primers for the genes of interest
  34. ES culture media (see Recipes)

Equipment

  1. Sterile cell culture hood (Safemate Vision 1.2 cabinet, catalog number: LDE0820)
  2. 37 °C cell culture incubator with 10% CO2 and 5% O2 (hypoxia) (Thermo Fisher Scientific, model: HeracellTM 150)
  3. Automated cell counter (NanoEnTek, catalog number: E1000)
  4. Refrigerated microcentrifuge (Bio-strategy, catalog number: 75002421)
  5. qPCR machine (7500 Real-Time PCR System) (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4351105)
  6. Vortexer
  7. Freezer

Software

  1. SDSv2.4 (Thermo Fisher Scientific, www.thermofisher.com/au/en/home/technical-resources/software-downloads/applied-biosystems-7900ht-fast-real-timespcr-system.html)
  2. GraphPad Prism 7 (GraphPad Sowtware, Inc, www.graphpad.com)
  3. Microsoft Excel Version 15.41 (Microsoft)

Procedure

  1. Cell culture and sample generation
    1. Seed 3 x 105 cells per well in 3 ml of media in each well of a 6-well plate (Figure 1–step 1, in total 6 wells per replicate).
      Note: We used mouse induced pluripotent stem cells (iPS cells) generated from reprogrammable tamoxifen inducible SRSF3-knockout and control mouse embryonic fibroblasts (MEFs) (Ratnadiwakara et al., 2018). The culture conditions described here reflect the requirements for these cells. A detailed description of the generation and culture properties of the iPS cell lines used here can be found in (Ratnadiwakara et al., 2018) and the media composition can be found under Recipes. This protocol is applicable to a wide range of tissue culture cell lines and the culture properties should be adjusted depending on the cell line used. 
    2. Let the cells adhere to the culture dish for 4 h, after which treat the cells with 5 µM tamoxifen (4OHT) to induce Cre-activity and SRSF3 depletion (Figure 1–step 2).
      Note: We have tested a range of tamoxifen (4OHT) concentrations and 5 µM 4OHT results in high recombination efficiency with minimal cytotoxicity.
    3. After 24 h, collect the cells from the first well as the first-time point by brief trypsination (0.1% trypsin for 3 min at 37 °C) or by using a cell scraper; t = 0 (Figure 1–step 3). A rapid processing of samples is required for the accuracy of the time points.
    4. Spin down the collected cells at 470 x g for 3 min at 20 °C. 
    5. Re-suspend the cell pellet in 1 ml of TRI Reagent and freeze at -80 °C. 
    6. To the remaining 5 wells, add 30 µl of 1 mg/ml Actinomycin D stock to obtain a final concentration of 10 µg/ml in 3 ml of culture media (Figure 1–step 3).
      Note: Make 1 mg/ml Actinomycin D stock in DMSO and freeze in aliquots at -20 °C. Dilute 30 µl of Actinomycin D stock in 100 µl of media and add dropwise to each well for uniform distribution.
    7. Collect samples at 1, 2, 4, 6 and 8 h time points following Actinomycin D addition and freeze the cell pellet in 1 ml of TRI Reagent as described above (Figure 1–steps 4 and 5).


      Figure 1. Experimental setup and sample collection. Collect the samples at relevant time points and proceed to RNA extraction. 

  2. RNA extraction
    1. Thaw the cells frozen in TRI Reagent at room temperature for 10 min. 
    2. Add 200 µl of chloroform and shake vigorously for 15 s and allow to stand for 10 min at room temperature.
    3. Centrifuge at 12,000 x g for 15 min at 4 °C.
    4. Transfer the aqueous layer into a fresh tube and add 500 µl of 2-propanol and 1 µl of Glycogen.
      Note: Addition of glycogen helps the maximum recovery of the RNA and visualization of the RNA pellet. 
    5. Mix carefully and allow to stand at room temperature for 10 min to precipitate the RNA.
    6. Centrifuge the mixture at 12,000 x g for 10 min at 4 °C.
    7. Remove the supernatant and wash the RNA pellet by adding 1 ml of 75% ethanol, vortex and centrifuge at 7,500 x g for 5 min at 4 °C.
    8. Briefly air-dry the RNA pellet for 5-10 min.
      Note: Avoid over-drying the pellet as this will greatly decrease its solubility.
    9. Re-suspend RNA in 30 µl of RNase-free water and proceed to cDNA Synthesis. 

  3. Reverse transcription and quantitative real-time PCR
    1. Perform DNaseI treatment on all samples to remove potential genomic DNA contamination that can affect the downstream analysis. We used Promega RQ1 DNase kit according to the manufacturer’s protocol and use 1 µg of RNA per sample.
    2. Carry out the reverse transcription of the RNA samples using a reverse transcription kit. We used SuperScript III Reverse transcriptase according to the manufacture’s protocol with equal amounts of random hexamers and oligodT18 for reverse priming. In short, 1 µg of DNaseI-treated RNA is incubated with dNTPs and primers at 65 °C for 5 min, followed by the addition of SuperScript III reagents. The cDNA synthesis is performed at 50 °C for 1 h and the reaction terminated by incubation at 70 °C for 15 min.
      Note: A mixture of random hexamers and oligodT18 can improve the sensitivity of the cDNA synthesis.
    3. Dilute the cDNA 1:10 with nuclease-free water to be used as a template for quantitative PCR. 
    4. Perform quantitative PCR with the primers specific for the gene of interest. Use the optimized manufacturer’s protocol for specific SYBR Green master mixes. We used 5 µl of Luminaries 2x HiGreen qPCR Master Mix per reaction, optimized concentration of each forward and reverse primers (typically 0.15-0.30 µM end concentration) and 2 µl of diluted cDNA per reaction. Perform two or more technical replicates for each sample.

Data analysis

  1. Upon the completion of the PCR, run the machine specific software to extract the data. We used SDS software.
    Note: The dissociation curve should produce a single peak for each reaction indicating the amplification of a single specific product (Figure 2C).
  2. Export Ct values to Excel spread sheet and calculate the average of replicates for each reaction. 
  3. Normalize the Ct average of each time point to the Ct average value of t = 0 to obtain ∆Ct value.
    ∆Ct = (Average Ct of each time point - Average Ct of t=0).
  4. Calculate the relative abundance for each time point.
    mRNA abundance = 2(-∆CT)
  5. Plot the relative abundance of mRNA at each time point relative to t = 0 using GraphPad Prism or similar software (Figures 2A and 2D). 
  6. Determine the mRNA decay rate by non-linear regression curve fitting (one phase decay) using GraphPad Prism (Figure 2B). We used following parameters:
    1. Least squares (ordinary fit)
    2. Confidence level–95%
    3. Asymmetrical (likelihood) CI
    4. Goodness of fit was quantified with R square
    5. Convergence criteria–medium
  7. This protocol describes one biological replicate but at least three independent experiments should be performed for statistical assessment.


    Figure 2. Data analysis. A. Relative Myc mRNA abundance (2(-∆CT)) for each time point for 2 replicates in control and SRSF3 depleted iPS cell samples. B. One phase decay analysis in GraphPad Prism. C. The dissociation curve with a single peak indicating the amplification of a single PCR product. D. Graph representing Myc mRNA decay in control and SRSF3-knockout (KO) cells after Actinomycin D treatment demonstrating similar half-lives for Myc mRNA in control and SRSF3-KO cells.

Recipes

  1. ES culture media
    1x Knock-Out DMEM
    15% ESC grade fetal bovine serum
    2 mM GlutaMAX
    1 mM Non-essential amino acids
    1x Penicillin-streptomycin
    Add 1,000 U/ml Leukemia Inhibitory Factor (LIF) and 1 µM beta-mercaptoethanol just before use

Acknowledgments

MLA was supported by National Health and Medical Research Council (NHMRC) GNT1043092 and GNT1138870, Aatos and Jane Erkko Foundation and Monash Biomedicine Discovery Fellowship.

Competing interests

The authors have no conflicts of interest or competing interests.

References

  1. Anko, M. L., Muller-McNicoll, M., Brandl, H., Curk, T., Gorup, C., Henry, I., Ule, J. and Neugebauer, K. M. (2012). The RNA-binding landscapes of two SR proteins reveal unique functions and binding to diverse RNA classes. Genome Biol 13(3): R17.
  2. Avendano, C. and J. Menéndez (2008). DNA Intercalators and topoisomerase inhibitors. Medicinal Chemistry of Anticancer Drugs. Elsevier Inc.: Madrid, Spain: 199-228.
  3. Bensaude, O. (2011). Inhibiting eukaryotic transcription: Which compound to choose? How to evaluate its activity? Transcription 2(3): 103-108.
  4. Berg J. M., Tymoczko J. L. and Stryer L. (2002). Transcription Is Catalyzed by RNA Polymerase. In: Biochemistry. 5th edition. New York: W H Freeman and Company. 
  5. Chen, C. Y., Ezzeddine, N. and Shyu, A. B. (2008). Messenger RNA half-life measurements in mammalian cells. Methods Enzymol 448: 335-357. 
  6. Harrold, S., Genovese, C., Kobrin, B., Morrison, S. L. and Milcarek, C. (1991). A comparison of apparent mRNA half-life using kinetic labeling techniques vs decay following administration of transcriptional inhibitors. Anal Biochem 198(1): 19-29.
  7. Ratnadiwakara, M., Archer, S. K., Dent, C. I., Ruiz De Los Mozos, I., Beilharz, T. H., Knaupp, A. S., Nefzger, C. M., Polo, J. M. and Anko, M. L. (2018). SRSF3 promotes pluripotency through Nanog mRNA export and coordination of the pluripotency gene expression program. Elife 7: e37419.
  8. Shyu, A. B., Greenberg, M. E. and Belasco, J. G. (1989). The c-fos transcript is targeted for rapid decay by two distinct mRNA degradation pathways. Genes Dev 3(1): 60-72.

简介

通过转录和转录后水平的多个步骤调节基因表达。细胞中成熟mRNA种类的净丰度由转录和降解之间的平衡决定。因此,mRNA稳定性的调节是关键的转录后事件,其可以极大地影响细胞中mRNA的净水平。可以通过分析转录抑制后的mRNA半衰期间接测量细胞内的mRNA稳定性,其中假定mRNA水平的变化反映mRNA降解。确定mRNA半衰期作为mRNA稳定性的量度可用于理解基因表达变化和调节不同生理条件或发育阶段的转录物水平的潜在机制。这里描述的方案提供了mRNA衰变的分析,作为在对照和SRSF3耗尽的小鼠诱导的多能干细胞(iPSC)中用放线菌素D处理转录抑制后测定mRNA稳定性的量度。

【背景】 确定细胞内mRNA的稳定性为理解转录后基因调控和RNA-蛋白质相互作用在该过程中的潜在作用提供了重要的措施。在任何特定条件下,例如跟随细胞外刺激或基因敲除,mRNA的稳定性可能由于增强的降解或延长的半衰期而改变(Shyu 等,,1989)。因此,为了评估mRNA稳定性,已经以多种方式直接测量内源mRNA的衰变速率,包括动力学标记技术和转录抑制剂的使用(Chen 等,,2008)。测量mRNA稳定性的最简单技术之一是通过用转录抑制剂抑制体内转录并测量mRNA动力学。

放线菌素D是转录抑制剂,其嵌入DNA中。放线菌素D与DNA形成非常稳定的复合物,防止DNA双螺旋的解旋,从而抑制DNA依赖性RNA聚合酶活性。放线菌素D广泛用于mRNA稳定性测定以抑制新mRNA的合成,允许通过测量转录抑制后的mRNA丰度来评估mRNA衰变(Avendano和Menéndez,2008)。在低浓度下,放线菌素D抑制转录而不显着影响DNA复制或蛋白质合成(Berg et al。,2002)。其他转录抑制剂如5,6-二氯-1β-1-呋喃核糖基苯并咪唑(DRB)与RNA聚合酶II直接相互作用也已成功用于类似的检测(Harrold et al。,1991) 。有关可以使用的不同转录抑制剂及其特定属性的更多详细信息,请参阅Bensaude(2011)。更先进的技术,如使用诱导型启动子控制瞬时转录,已经显示出优于放线菌素D或其他转录抑制剂在mRNA衰变分析中的潜在细胞毒性作用(Chen et al。,2008) 。然而,放线菌素D检测的优势在于它不需要构建和将外源基因导入细胞,并提供了一种测量内源mRNA稳定性变化的方法(Chen et al。,2008) 。

我们已经建立了富含丝氨酸 - 精氨酸的剪接因子3(SRSF3)-RNA相互作用,作为协调多能细胞中基因表达的关键手段(Ratnadiwakara et al。,2018)。此外,据报道SRSF3通过与无义介导的衰变(NMD)偶联的可变剪接来调节mRNA水平,包括其自身的mRNA丰度(Anko 等,,2012)。为了确定SRSF3是否影响多能细胞中NMD敏感性转录物变体的产生或稳定性,我们测定了放线菌素D处理的对照和SRSF3耗尽的多能干细胞中SRSF3靶mRNA的mRNA半衰期(Ratnadiwakara 等。 / em>,2018)。几种技术如Northern印迹分析,原位杂交和定量PCR可用于确定转录抑制后的mRNA半衰期。我们使用定量PCR,可以快速,灵敏地测量各种表达水平的mRNA半衰期,包括低丰度的mRNA。此处描述的方案可用于成功测量多能干细胞中的mRNA衰变。

关键字:mRNA稳定性, 放线菌素 D, mRNA降解, 转录抑制剂, mRNA半衰期

材料和试剂

  1. 6孔细胞培养板(Sigma-Aldrich,目录号:CLS3516)
  2. 血清移液器10 ml(Sigma-Aldrich,目录号:CLS4488)
  3. 15毫升Falcon管(Sigma-Aldrich,目录号:CLS430791)
  4. 无菌过滤器移液器吸头10μl,20μl,200μl,1,000μl(Axygen,目录号:TF10LRS,TF20LRS,TF200LRS和TF1000LRS)
  5. 微量离心管(Axygen,目录号:MCT-175-C)
  6. 海王星半裙96孔板(VWR,目录号:89126-694)
  7. 光学透明粘合剂密封片(Thermo Fisher Scientific,目录号:AB-1170)
  8. 敲除DMEM(Thermo Fisher Scientific,目录号:10829018)
  9. ES细胞级胎牛血清(Sigma-Aldrich,目录号:F9423)
  10. GlutaMAX(Life Technologies,目录号:35050-061)
  11. 胰蛋白酶0.25%(Life Technologies,目录号:25200-056)
  12. 青霉素 - 链霉素(Life Technologies,目录号:15070-063)
  13. 非必需氨基酸(Life Technologies,目录号:11140-050)
  14. Beta-Mercapto乙醇(生命技术,目录号:21985-023)
  15. 白血病抑制因子(LIF)(此处LIF由澳大利亚蒙纳士大学蛋白质生产部门生产)
  16. 磷酸盐缓冲盐水(PBS)(Life Technologies,目录号:14190-250)
  17. 培养细胞(这里是小鼠诱导的多能干细胞)
  18. (z)-4-羟基三苯氧胺(Sigma-Aldrich,目录号:H7904-5MG)
  19. 细胞培养级放线菌素D(Sigma-Aldrich,目录号:A9415) 
  20. 细胞培养级二甲基亚砜(DMSO)(AppliChem,目录号:A3672,0100) 
  21. TRI Reagent(西格玛奥德里奇,目录号:T9424)
  22. 氯仿(Sigma-Aldrich,目录号:288306)
  23. 异丙醇(Sigma-Aldrich,目录号:278475)
  24. RNA级糖原(Thermo Fisher Scientific,目录号:R0551)
  25. 乙醇(任何分子级)
  26. 无RNase水(Invitrogen,目录号:10977-015) 
  27. RQ1 DNase试剂盒(Promega,目录号:M6101)
  28. SuperScript III逆转录试剂盒(Thermo Fisher Scientific,目录号:18080044)
  29. RNaseOUT(赛默飞世尔科技,目录号:10777019)
  30. 随机六聚体底漆混合物(Bioline,目录号:BIIO38028)
  31. OligodT 18 (IDT)
  32. SYBR green master mix,这里是Luminaries HiGreen qPCR Master Mix,Low ROX(赛默飞世尔科技,目录号:K0974)
  33. 用于感兴趣基因的qPCR引物
  34. ES文化媒体(见食谱)

设备

  1. 无菌细胞培养罩(Safemate Vision 1.2柜,目录号:LDE0820)
  2. 37°C细胞培养箱,含10%CO 2 和5%O 2 (缺氧)(Thermo Fisher Scientific,型号:Heracell TM 150 )
  3. 自动细胞计数器(NanoEnTek,目录号:E1000)
  4. 冷冻微量离心机(Bio-strategy,目录号:75002421)
  5. qPCR机器(7500实时PCR系统)(Thermo Fisher Scientific,Applied Biosystems TM ,目录号:4351105)
  6. 涡流混合器
  7. 冰箱

软件

  1. SDSv2.4(赛默飞世尔科技, www.thermofisher.com/au/en/home/technical-resources/software-downloads/applied-biosystems-7900ht-fast-real-timespcr-system.html
  2. GraphPad Prism 7(GraphPad Sowtware,Inc, www.graphpad.com
  3. Microsoft Excel版本15.41(Microsoft)

程序

  1. 细胞培养和样品生成
    1. 在6孔板的每个孔中的3ml培养基中每孔接种3×10 5个/×sup细胞(图1-步骤1,每个重复6个孔)。
      注意:我们使用由可重编程的他莫昔芬诱导型SRSF3敲除和对照小鼠胚胎成纤维细胞(MEF)产生的小鼠诱导的多能干细胞(iPS细胞)(Ratnadiwakara等,2018)。这里描述的培养条件反映了这些细胞的要求。这里使用的iPS细胞系的产生和培养特性的详细描述可以在(Ratnadiwakara等,2018)中找到,并且培养基组合物可以在食谱下找到。该方案适用于广泛的组织培养细胞系,培养特性应根据使用的细胞系进行调整。
    2. 让细胞粘附在培养皿上4小时,然后用5μM他莫昔芬(4OHT)处理细胞以诱导Cre活性和SRSF3耗尽(图1步骤2)。
      注意:我们测试了一系列他莫昔芬(4OHT)浓度和5μM4OHT,导致重组效率高,细胞毒性最小。
    3. 24小时后,通过短暂的胰蛋白酶消化(0.1%胰蛋白酶在37℃下3分钟)或使用细胞刮刀收集第一个孔中的细胞作为第一个时间点; t = 0(图1 - 步骤3)。为了时间点的准确性,需要快速处理样本。
    4. 将收集的细胞在470℃下旋转,在20℃下旋转3分钟。 
    5. 将细胞沉淀重悬于1ml TRI试剂中并在-80℃下冷冻。 
    6. 向剩余的5个孔中加入30μl1mg / ml放线菌素D原液,在3 ml培养基中获得10μg/ ml的终浓度(图1 - 步骤3)。
      注意:在DMSO中制备1 mg / ml放线菌素D原液,并在-20°C下等分冷冻。在100μl培养基中稀释30μl放线菌素D原液,逐滴添加到每个孔中均匀分布。
    7. 在添加放线菌素D后的1,2,4,6和8小时时间点收集样品,并如上所述将细胞沉淀冷冻在1ml TRI试剂中(图1-步骤4和5)。


      图1.实验设置和样品采集。在相关时间点收集样品并进行RNA提取。 

  2. RNA提取
    1. 将在室温下在TRI试剂中冷冻的细胞解冻10分钟。 
    2. 加入200μl氯仿,剧烈振荡15秒,在室温下静置10分钟。
    3. 在4℃下以12,000 x g 离心15分钟。
    4. 将水层转移到新管中,加入500μl2-丙醇和1μl糖原。
      注意:添加糖原有助于RNA的最大恢复和RNA颗粒的可视化。 
    5. 小心混合并在室温下静置10分钟以沉淀RNA。
    6. 在4℃下将混合物在12,000 x g 下离心10分钟。
    7. 除去上清液并通过加入1ml 75%乙醇洗涤RNA沉淀,涡旋并在4,500℃离心7分钟,在4℃下离心5分钟。
    8. 将RNA颗粒短暂风干5-10分钟。
      注意:避免过度干燥颗粒,因为这会大大降低其溶解度。
    9. 将RNA重悬于30μl不含RNase的水中,然后进行cDNA合成。 

  3. 逆转录和定量实时PCR
    1. 对所有样品进行DNaseI处理,以去除可能影响下游分析的潜在基因组DNA污染。我们根据制造商的方案使用Promega RQ1 DNase试剂盒,每个样品使用1μgRNA。
    2. 使用逆转录试剂盒进行RNA样品的逆转录。我们根据制造商的方案使用SuperScript III Reverse transcriptase,使用等量的随机六聚体和oligodT 18 进行反向引发。简而言之,将1μg经DNaseI处理的RNA与dNTP和引物在65℃温育5分钟,然后加入SuperScript III试剂。 cDNA合成在50℃下进行1小时,通过在70℃温育15分钟终止反应 注意:随机六聚体和oligodT 18 的混合物可以提高cDNA合成的灵敏度。
    3. 用不含核酸酶的水稀释cDNA 1:10,作为定量PCR的模板。 
    4. 用对目的基因特异的引物进行定量PCR。将优化的制造商协议用于特定的SYBR Green主混合物。我们每次反应使用5μlLuminaries2x HiGreen qPCR Master Mix,优化每种正向和反向引物的浓度(通常0.15-0.30μM终浓度)和每次反应2μl稀释的cDNA。对每个样品执行两个或多个技术重复。

数据分析

  1. 完成PCR后,运行机器专用软件以提取数据。我们使用SDS软件。
    注意:解离曲线应为每个反应产生一个峰,表明单个特定产物的扩增(图2C)。
  2. 将Ct值导出到Excel电子表格并计算每个反应的重复平均值。 
  3. 将每个时间点的Ct平均值归一化为Tt 0的Ct平均值,以获得ΔCt值。
    ΔCt=(每个时间点的平均Ct - t = 0的平均Ct)。
  4. 计算每个时间点的相对丰度。
    mRNA丰度= 2(-ΔCT)
  5. 使用GraphPad Prism或类似软件绘制相对于t = 0的每个时间点的mRNA的相对丰度(图2A和2D)。 
  6. 使用GraphPad Prism通过非线性回归曲线拟合(一相衰减)确定mRNA衰减速率(图2B)。我们使用以下参数:
    1. 最小二乘(普通拟合)
    2. 置信水平-95%
    3. 不对称(可能性)CI
    4. 用R square量化拟合优度
    5. 收敛标准 - 中等
  7. 该协议描述了一个生物学重复,但至少应进行三次独立实验以进行统计学评估。


    图2.数据分析。 :一种。对于对照和SRSF3耗尽的iPS细胞样品中的2个重复的每个时间点的相对 Myc mRNA丰度(2(-ΔCT))。 B. GraphPad Prism中的一相衰减分析。 C.具有单峰的解离曲线表明单个PCR产物的扩增。 D.代表放线菌素D处理后对照和SRSF3敲除(KO)细胞中 Myc mRNA衰变的图表,显示对照和SRSF3-KO细胞中 Myc mRNA的相似半衰期。

食谱

  1. ES文化媒体
    1x淘汰DMEM
    15%ESC级胎牛血清
    2 mM GlutaMAX
    1 mM非必需氨基酸
    1x青霉素 - 链霉素
    使用前加入1,000 U / ml白血病抑制因子(LIF)和1μMβ-巯基乙醇

致谢

MLA得到了国家健康与医学研究委员会(NHMRC)GNT1043092和GNT1138870,Aatos和Jane Erkko基金会以及Monash Biomedicine Discovery Fellowship的支持。

竞争利益

作者没有利益冲突或利益冲突。

参考

  1. Anko,M.L.,Muller-McNicoll,M.,Brandl,H.,Curk,T.,Gorup,C.,Henry,I.,Ule,J。和Neugebauer,K.M。(2012)。 两种SR蛋白的RNA结合区域揭示了独特的功能并与不同的RNA类别结合。 Genome Biol 13(3):R17。
  2. Avendano,C。和J.Menéndez(2008)。 DNA嵌入剂和拓扑异构酶抑制剂。 抗癌药物的药物化学。 Elsevier公司:西班牙马德里:199-228。
  3. Bensaude,O。(2011)。 抑制真核转录:选择哪种化合物?如何评估其活动? 转录 2(3):103-108。
  4. Berg J. M.,Tymoczko J. L.和Stryer L.(2002)。 RNA聚合酶催化转录。在:生物化学。第5版。纽约:W H Freeman and Company。 
  5. Chen,C。Y.,Ezzeddine,N。和Shyu,A。B.(2008)。 在哺乳动物细胞中进行信使RNA半衰期测量。 Methods Enzymol 448:335-357。 
  6. Harrold,S.,Genovese,C.,Kobrin,B.,Morrison,S.L。和Milcarek,C。(1991)。 使用动力学标记技术比较表观mRNA半衰期与使用转录抑制剂后的衰变进行比较。 / a> Anal Biochem 198(1):19-29。
  7. Ratnadiwakara,M.,Archer,S.K.,Dent,C.I.,Ruiz De Los Mozos,I.,Beilharz,T.H.,Knaupp,A.S.,Nefzger,C.M.,Polo,J.M。和Anko,M.L。(2018)。 SRSF3通过 Nanog mRNA输出和多能性基因表达的协调来促进多能性程序。 Elife 7:e37419。
  8. Shyu,A.B.,Greenberg,M。E.和Belasco,J.G。(1989)。 c-fos转录物的目标是通过两种不同的mRNA降解途径快速衰变。 Genes Dev 3(1):60-72。
  • 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 Ratnadiwakara and Änkö. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Ratnadiwakara, M. and Änkö, M. (2018). mRNA Stability Assay Using Transcription Inhibition by Actinomycin D in Mouse Pluripotent Stem Cells. Bio-protocol 8(21): e3072. DOI: 10.21769/BioProtoc.3072.
  2. Ratnadiwakara, M., Archer, S. K., Dent, C. I., Ruiz De Los Mozos, I., Beilharz, T. H., Knaupp, A. S., Nefzger, C. M., Polo, J. M. and Anko, M. L. (2018). SRSF3 promotes pluripotency through Nanog mRNA export and coordination of the pluripotency gene expression program. Elife 7: e37419.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。