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Measurement of mRNA Decay in Mouse Embryonic Fibroblasts
小鼠胚胎成纤维细胞中mRNA Decay的测量   

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Abstract

mRNA stability control is a critical step in the post-transcriptional regulation of gene expression. Actinomycin D, an antibiotic initially used as an anti-cancer drug, has turned out to be a convenient tool for studying the turnover rates of transcripts in cells, due to its inhibition of mRNA synthesis. Here, we describe a protocol for the measurement of mRNA decay after adding actinomycin D into the medium of stable fibroblast cell lines derived from wild-type and tristetraprolin (TTP)-deficient mouse embryonic fibroblast (MEF) cultures, as well as a protocol for determining the relative transcript abundance using semi-quantitative real time RT-PCR. Northern blotting or NanoString n-Counter are alternative methods to measure mRNA abundance, which is quantified using a phosphorimager in the former case. This protocol is suitable for studying primary cultured cells and stable cell lines derived from transgenic mice and their respective controls, and provides for direct comparisons of mRNA decay rates in otherwise identical cells with and without the gene of interest.

Keywords: MRNA decay(mRNA的衰变), Mouse embryonic fibroblasts(小鼠胚胎成纤维细胞), Actinomycin D(actinomycin D), Real-time RT-PCR(实时RT-PCR)

Materials and Reagents

  1. 60-mm sterile petri dish (e.g., BD Biosciences, Falcon®, catalog number: 353002 )
    Note: Currently, it is “Corning, Falcon®, catalog number: 353002”.
  2. T-75 tissue culture flask (e.g., BD Biosciences, Falcon®, catalog number: 353136 )
    Note: Currently, it is “Corning, Falcon®, catalog number: 353136”.
  3. 50 ml sterile conical tube (e.g., BD Biosciences, Falcon®, catalog number: 352070 )
    Note: Currently, it is “Corning, Falcon®, catalog number: 352070”.
  4. 384-well microplate (e.g., BioExpress, catalog number: T-6062-1 )
  5. 1.7 ml RNase-free, DNase-free Posi-Click tubes (Denville Scientific Inc., catalog number: C2170 )
  6. Mouse wild-type (WT) and TTP-deficient stable fibroblast cell lines (Lai WS et al., 2006)
  7. 1x Phosphate-buffered saline (PBS) without calcium and magnesium
  8. 0.05% trypsin/EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25300 )
  9. Fetal bovine serum defined (FBS) (GE Healthcare, HyCloneTM, catalog number: SH30070.03 )
  10. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11965-092 )
  11. Penicillin-Streptomycin 10,000 U/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  12. L-glutamine 200 mM (Thermo Fisher Scientific, GibcoTM, catalog number: 25030-081 )
  13. Recombinant mouse tumor necrosis factor (TNF) (R&D Systems, catalog number: 410-MT )
  14. Actinomycin D (Sigma-Aldrich, catalog number: A4262 )
  15. Illustra RNAspin MiniRNA isolation kit (Sigma-Aldrich, GE Healthcare, catalog number: 25-0500-72 )
  16. SuperScript First-Strand Synthesis System (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18080-051 )
  17. Power SYBR Green master mix (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4368702 )
  18. Mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  19. DEPC water (Baltimore Bioworks, catalog number: WA-137-500 )
  20. Primers for transcripts of interest
  21. 70% ethanol
  22. Complete medium (see Recipes)
  23. Serum-starving medium (see Recipes)

Equipment

  1. 37 °C, 5% CO2 forced-air incubator (e.g., Thermo Fisher Scientific, FormaTM, model: 3110 )
  2. Centrifuge with swinging-bucket rotor and adaptors for 50-ml conical tubes
  3. ABI Prism 7900HT Real-Time PCR System and Sequence Detection System (Applied Biosystems, model: 7900HT) or similar
  4. Vortex-Genie-2 (Scientific Industries, catalog number: SI-0236 ) or similar
  5. Nanodrop 2000c spectrophotometer (Thermo Scientific, model: 2000c)
  6. Desktop centrifuge (e.g., Eppendorf, model: 5417R )
  7. DNA Engine Peltier Thermal Cycler (Bio-Rad Laboratories, MJ research, catalog number: PTC-200 ) or similar
  8. Cell scraper (Corning, Costar, catalog number: 3010 )

Software

  1. GraphPad Prism software (GraphPad Software, model: version 6.0)

Procedures

  1. Cell culture
    1. Mouse stable fibroblast cell lines were derived from MEF cultures from E14.5 TTP KO and littermate WT embryos, as described previously (Lai WS et al., 2006). These stable cell lines have been cultured for more than 200 passages and are well matched in terms of growth rates, morphology, and responses of rapidly inducible genes, such as Fos, to serum stimulation.
    2. The two stable fibroblast cell lines are maintained in complete medium in T-75 flasks, and passaged every 2-3 days after achieving approximate 70-80% confluence and trypsin treatment.
      Note: Cell culture, stimulation and actinomycin D treatment are performed in a Class II Biological Safety Cabinet.

  2. Serum starvation and stimulation
    1. To plate the cells for experiments, cells are harvested from 3-5 T-75 flasks after washing with 10 ml of PBS followed by trypsinization with 2 ml of trypsin/EDTA and neutralization with 8 ml of complete medium per one T-75 flask, and plated into 60-mm petri dishes at a density of 2-3 x 105 cells in 5 ml culture medium per petri dish.
      Note: The estimated cell counts harvested from each T-75 flask range between 8 x 105 and 3 x 106 for both the 66 KO and 67 WT stable cell lines, with an average yield of approximate 1.4 x 106 cells.
    2. When the cells reach 70-80% confluence, the cells are washed in serum-free DMEM and then cultured in 5 ml serum-starving medium for at least 16 h of serum starvation.
    3. Add recombinant murine TNF (or other stimuli) into serum-starving medium for a final TNF concentration of 10 ng/ml, and then harvest cells at various time points after treatment.

  3. Actinomycin D treatment
    1. Actinomycin D solution is prepared by dissolving the powder in DEPC water. The solution is stored at 4 °C in the dark for a final concentration of 2-5 mg/ml.
      Note: It often takes at least 1-2 days for the actinomycin D to get fully dissolved in water at 4 °C; gently invert the bottle several times to mix the solution prior to use. DMSO is not used as a solvent in this protocol.
    2. After 30 min or other times of TNF treatment of fibroblast cells (10 ng/ml in this example), or treatment with other stimuli, actinomycin D is added directly to the serum-starving medium for a final concentration of 5-10 μg/ml; TNF and other stimuli are not removed at the time of actinomycin D addition.
      Note: We routinely perform a time course study first for an individual stimulus to determine the kinetic expression pattern of the gene of interest. The time point at which cells express the highest levels of the transcript of interest is then usually chosen as the time for actinomycin D addition.
    3. Cells are then harvested at 0, 10, 20, 30, 45, 60, 90, and 120 min after the addition of actinomycin D, or other times as indicated; each sample is comprised of pooled cells from three 60-mm dishes.
      Note: It is not recommended to use trypsin/EDTA for cell harvest followed by RNA extraction. It is necessary to proceed immediately to Procedure D by adding sample lysis buffer directly into the petri dishes after one rinse with ice-cold PBS.

  4. RNA extraction, reverse transcription and semi-quantitative real-time PCR
    1. To prepare cells for RNA extraction, the culture medium is aspirated and the cells are washed once in 5 ml of ice-cold PBS per one 60-mm dish. RA1 lysis buffer (from the Illustra RNAspin MiniRNA isolation kit) supplemented with freshly added mercaptoethanol (ME, 1:100 dilution) is added directly into the petri dishes, and the lysates are then scraped off using cell scrapers. The lysates are then pooled from triplicate plates and transferred into a new fresh 1.7 ml microcentrifuge tube. This procedure can be performed on the bench at room temperature.
      Note: For cells in suspension other than adherent cells such as fibroblasts, samples are harvested first by centrifugation, and then washed once in PBS before adding RA1 lysis buffer with ME.
    2. Follow the manufacturer’s instructions in the GE Healthcare illustra RNAspin MiniRNA isolation kit for total RNA extraction; this includes steps of lysate vortexing, filtering through the shredder column to decrease the viscosity of lysates, and on-column digestion with RNase-free DNase I. Lysates that have flowed through the shredder column can be transferred into a new tube and stored at -80 °C.
    3. Take 1 μl of RNA from each sample to check for RNA quantity and quality with Nanodrop.
      Note: The expected RNA yield from three combined dishes in this protocol is around 16 µg.
    4. 750 ng RNA is used to synthesize first-strand cDNAs using oligo (dT)12-18 primers and SuperScript III Reverse Transcriptase (Invitrogen) as per manufacturer’s protocol.
    5. cDNA is then diluted to 1.5-2 ng/µl with DEPC water, and is ready for use or can be stored at -20 °C.
    6. Real-time PCR is performed using SYBR Green master mix and the ABI Prism 7900 Sequence Detection System in a 384-well plate. Each reaction is comprised of 1x SYBR Green master mix, 1-2 ng cDNA, 250 nM of each primer in a total of 10 μl reaction volume, i.e., 5 µl of 2x SYBR Green master mix, 0.5 µl of each forward and reverse primer at 5 µM, 1 µl of cDNA, and 3 µl of DEPC water per reaction. Each plate contains “no template” controls for individual transcripts as well as housekeeping transcripts such as Actb mRNA for every sample as an internal control.
    7. Results are analyzed using the ΔΔCt method (Pfaffl MW 2001). Ct values from duplicate or triplicate samples are first normalized to their respective internal housekeeping transcripts, Actb mRNA in this example, and then normalized to their respective samples before the addition of actinomycin D, which are set at 1. The results are expressed as percentages of mRNA abundance relative to time 0. A representative experiment is shown in Table 1.
      Note: It is critical to validate primer amplification efficiency and specificity prior to use. To check primer amplification efficiency, a 10-fold serial dilution of cDNA across 4-5 log range is used. The relative Ct values after normalization to an internal housekeeping transcript are plotted against the concentrations at log values for linear regression curves. Any absolute values of slopes less than or equal to 0.1 are considered to pass the efficiency test when paired with the housekeeping gene in the assay. A representative experiment is shown in Figure 1.

      Table 1. Calculation of the Relative Transcript Abundance using the ΔΔCt Method

      *∆Ct = Ct (Fos, column 2) – Ave Ct (Actb, column 4); **∆∆Ct = ∆Ct (column 5)– Ave ∆Ct at time 0 (column 6). Actinomycin D (ActD) was added after 30 min of TNF stimulation.

    8. The transcript turnover rates are then calculated based on the non-linear fit one phase exponential decay curves using GraphPad Prism software, and are generally expressed as times to 50% mRNA decay for each experiment. A representative experiment is shown in Figure 2.

Representative data



Figure 1. Validation of real-time PCR primers. Serial dilutions of cDNA at 10, 5, 1, 0.5, 0.1, and 0.01 ng were prepared as templates for real-time PCR analysis using primers against Fos and Actb transcripts. The X-axis in the graph shown is the log value of cDNA; ∆Ct shown on the Y-axis are the differences in the Ct values between Fos and Actb mRNAs. The data presented are means ± SD of duplicates from one representative experiment. A linear regression curve was plotted with an absolute slope value of ≤0.1. In this example, the Ct value for the Fos transcript at 0.01 ng was undetectable, so it was not included in the calculation of the regression curve.


Figure 2. Stability of Fos and Cxcl1 transcripts after TNF stimulation in the presence and absence of TTP. Actinomycin D, to inhibit transcription, was added after 30 min of TNF treatment of serum-starved fibroblast cell lines. The cells were harvested for total RNA extraction at the indicated times, and mRNA levels were measured with real-time RT-PCR. Transcript concentrations were normalized to those of the Actb transcripts, and were expressed as fractions of abundance in the TNF-treated samples prior to the addition of actinomycin D. The results shown are means of replicate samples from one representative experiment. The transcript turnover rates were calculated based on the non-linear fit one phase exponential decay curves using GraphPad software (red dotted lines, 66 KO cells; blue dotted lines, 67 WT cells), and expressed as times to 50% mRNA decay for each experiment (the time points when the dotted black lines for the 50% mRNA decay lines crosses the red and blue dotted lines, respectively, for each curve).

Recipes

  1. Complete medium
    DMEM
    10% FBS
    1% Pen-Strep
    2 mM glutamine
  2. Serum-starving medium
    DMEM
    0.5% FBS
    1% Pen-Strep
    2 mM glutamine

Acknowledgments

This protocol was adapted from previously published studies, Lai et al. (2006) and Horner et al. (2009), and was used as described here in Qu et al. (2015). We thank Drs. Melissa Wells and Diana Cruz-Topete for comments on the protocol. This research was supported by the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health.

References

  1. Horner, T. J., Lai, W. S., Stumpo, D. J. and Blackshear, P. J. (2009). Stimulation of polo-like kinase 3 mRNA decay by tristetraprolin. Mol Cell Biol 29(8): 1999-2010.
  2. Lai, W. S., Parker, J. S., Grissom, S. F., Stumpo, D. J. and Blackshear, P. J. (2006). Novel mRNA targets for tristetraprolin (TTP) identified by global analysis of stabilized transcripts in TTP-deficient fibroblasts. Mol Cell Biol 26(24): 9196-9208.
  3. Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9): e45.
  4. Qiu, L. Q., Lai, W. S., Bradbury, A., Zeldin, D. C. and Blackshear, P. J. (2015). Tristetraprolin (TTP) coordinately regulates primary and secondary cellular responses to proinflammatory stimuli. J Leukoc Biol 97(4): 723-736.

简介

mRNA稳定性控制是基因表达的转录后调控中的关键步骤。放线菌素D,一种最初用作抗癌药物的抗生素,由于其对mRNA合成的抑制,已经证明是用于研究细胞中转录物的转换率的方便的工具。在这里,我们描述了添加放线菌素D后的稳定成纤维细胞细胞系,从野生型和三四氯丙胺(TTP)缺陷小鼠胚胎成纤维细胞(MEF)文化,以及一个协议使用半定量实时RT-PCR确定相对转录本丰度。 Northern印迹或NanoString n-Counter是测量mRNA丰度的备选方法,在前一种情况下使用phosphorimager对其进行定量。该方案适合于研究源自转基因小鼠及其各自对照的原代培养细胞和稳定细胞系,并且提供在具有和不具有目标基因的其他相同细胞中的mRNA衰减率的直接比较。

关键字:mRNA的衰变, 小鼠胚胎成纤维细胞, actinomycin D, 实时RT-PCR

材料和试剂

  1. 60-mm无菌培养皿(例如,BD Biosciences,Falcon ,目录号:353002)。
    注意:目前,"Corning,Falcon ? ,目录号:353002"。
  2. T-75组织培养瓶(例如BD Biosciences,Falcon ,目录号:353136)。
    注意:目前,它是"康宁,Falcon ? ,目录号:353136"。
  3. 50ml无菌锥形管(例如BD Biosciences,Falcon ,目录号:352070)。
    注意:目前,"Corning,Falcon ? ,目录号:352070"。
  4. 384孔微孔板(例如,BioExpress,目录号:T-6062-1)
  5. 1.7ml无RNase,无DNA酶的Posi-Click管(Denville Scientific Inc.,目录号:C2170)
  6. 小鼠野生型(WT)和TTP缺陷型稳定成纤维细胞细胞系(Lai WS等人,2006)
  7. 1x不含钙和镁的磷酸盐缓冲盐水(PBS)
  8. 0.05%胰蛋白酶/EDTA(Thermo Fisher Scientific,Gibco TM ,目录号:25300)
  9. 定义的胎牛血清(FBS)(GE Healthcare,HyClone ,目录号:SH30070.03)
  10. Dulbecco改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM ,目录号:11965-092)
  11. 青霉素 - 链霉素10,000U/ml(Thermo Fisher Scientific,Gibco< sup>,目录号:15140-122)
  12. L-谷氨酰胺200mM(Thermo Fisher Scientific,Gibco TM ,目录号:25030-081)
  13. 重组小鼠肿瘤坏死因子(TNF)(R& D Systems,目录号:410-MT)
  14. 放线菌素D(Sigma-Aldrich,目录号:A4262)
  15. Illustra RNAspin MiniRNA分离试剂盒(Sigma-Aldrich,GE Healthcare,目录号:25-0500-72)
  16. SuperScript First-Strand Synthesis System(Thermo Fisher Scientific,Invitrogen TM ,目录号:18080-051)
  17. Power SYBR Green master mix(Thermo Fisher Scientific,Applied Biosystems TM ,目录号:4368702)
  18. 巯基乙醇(Sigma-Aldrich,目录号:M3148)
  19. DEPC水(Baltimore Bioworks,目录号:WA-137-500)
  20. 感兴趣的抄本的入门
  21. 70%乙醇
  22. 完整介质(见配方)
  23. 血清饥饿培养基(见配方)

设备

  1. 37℃,5%CO 2强制空气培养箱(例如Thermo Fisher Scientific,Forma TM sup),型号:3110) >
  2. 带有摆动斗式转子的离心机和适用于50 ml锥形管的适配器
  3. ABI Prism 7900HT实时PCR系统和序列检测系统(Applied Biosystems,型号:7900HT)或类似物
  4. Vortex-Genie-2(Scientific Industries,目录号:SI-0236)或类似的
  5. Nanodrop 2000c分光光度计(Thermo Scientific,型号:2000c)
  6. 桌面离心机(,例如,Eppendorf,型号:5417R)
  7. DNA Engine Peltier热循环仪(Bio-Rad Laboratories,MJ research,目录号:PTC-200)或类似物
  8. 细胞刮刀(Corning,Costar,目录号:3010)

软件

  1. GraphPad Prism软件(GraphPad Software,型号:6.0版)

程序

  1. 细胞培养
    1. 如先前所述(Lai WS等人,2006),小鼠稳定的成纤维细胞系衍生自来自E14.5TTP KO和同窝出生的WT胚胎的MEF培养物。这些稳定的细胞系已经培养超过200代,并且在生长速率,形态学和快速诱导基因(例如Fos )对血清刺激的反应方面很好地匹配。
    2. 将两种稳定的成纤维细胞系保持在T-75烧瓶中的完全培养基中,并在达到约70-80%汇合和胰蛋白酶处理后每2-3天传代。
      注意:细胞培养,刺激和放线菌素D处理在II类生物安全柜中进行。

  2. 血清饥饿和刺激
    1. 为了将细胞平板用于实验,在用10ml PBS洗涤后,从3-5个T-75烧瓶中收获细胞,然后用2ml胰蛋白酶/EDTA胰蛋白酶化,并用每个T-75烧瓶中的8ml完全培养基中和,并在每个培养皿5ml培养基中以2-3×10 5个细胞的密度接种到60-mm培养皿中。
      注意:对于66KO和67WT稳定,从每个T-75瓶收获的估计的细胞计数范围为8×10 5至3×10 6个/细胞系,平均产量约为1.4×10 6个细胞。
    2. 当细胞达到70-80%汇合时,在无血清DMEM中洗涤细胞,然后在5ml血清饥饿培养基中培养至少16小时的血清饥饿。
    3. 将重组鼠TNF(或其他刺激物)加入血清饥饿培养基中,最终TNF浓度为10ng/ml,然后在处理后的不同时间点收集细胞。

  3. 放线菌素D处理
    1. 放线菌素D溶液通过将粉末溶解在DEPC水中制备。将溶液在4℃下在黑暗中储存,最终浓度为2-5mg/ml。
      注意:放线菌素D在4℃下完全溶解于水中通常需要至少1-2天;在使用前轻轻倒置瓶子几次以混合溶液。在本协议中,DMSO不用作溶剂。
    2. 在30分钟或其他时间的TNF处理成纤维细胞(在该实施例中为10ng/ml)或用其它刺激物处理后,将放线菌素D直接加入到血清饥饿培养基中,最终浓度为5-10μg/ml ; TNF和其他刺激在加入放线菌素D时不被除去。
      注意:我们常规地对单个刺激执行时间过程研究以确定感兴趣的基因的动力学表达模式。然后通常选择细胞表达感兴趣的转录物的最高水平的时间点作为放线菌素D加入的时间。
    3. 然后在加入放线菌素D后0,10,20,30,45,60,90和120分钟或如所示的其它时间收获细胞;每个样品由来自三个60-mm培养皿的合并细胞组成 注意:不建议使用胰蛋白酶/EDTA进行细胞收获,然后进行RNA提取。有必要立即进行程序D,在用冰冷的PBS冲洗后,将样品裂解缓冲液直接加入培养皿中。

  4. RNA提取,逆转录和半定量实时PCR
    1. 为了制备用于RNA提取的细胞,吸出培养基,并将细胞在每个60-mm培养皿中的5ml冰冷PBS中洗涤一次。将补充有新鲜添加的巯基乙醇(ME,1:100稀释)的RA1裂解缓冲液(来自Illustra RNAspin MiniRNA分离试剂盒)直接加入培养皿中,然后使用细胞刮刀刮下裂解物。然后从一式三份板中收集裂解物,并转移到新鲜的1.7ml微量离心管中。该程序可以在室温下在台上进行。
      注意:对于除了粘附细胞(例如成纤维细胞)以外的悬浮细胞,首先通过离心收集样品,然后在加入含ME的RA1裂解缓冲液之前在PBS中洗涤一次。
    2. 按照制造商在GE Healthcare illustra RNAspin MiniRNA分离试剂盒中的说明进行总RNA提取;这包括裂解物涡旋,通过切碎机柱过滤以降低裂解物的粘度,以及使用无RNA酶的DNase I进行柱上消化的步骤。已经流过切碎机柱的裂解物可以转移到新管中并储存在 - 80℃。
    3. 从每个样品中取1μl的RNA,以检查RNA的数量和质量与Nanodrop 注意:在该方案中来自三个组合培养皿的预期RNA产量为约16μg。
    4. 使用oligo(dT)Sub 12-18引物和SuperScript III Reverse Transcriptase(Invitrogen)根据制造商的方案使用750ng RNA合成第一链cDNA。
    5. 然后用DEPC水将cDNA稀释至1.5-2ng /μl,即可使用,或可在-20℃下保存。
    6. 使用SYBR Green主混合物和ABI Prism 7900序列检测系统在384孔板中进行实时PCR。每个反应由总共10μl反应体积的1x SYBR Green主混合物,1-2ng cDNA,250nM每种引物,5μl2x SYBR Green master mix,0.5μl μl的每种正向和反向引物(5μM),1μlcDNA和3μlDEPC水/反应。每个板含有单个转录物的"无模板"对照,以及每个样品作为内部对照的管理转录物例如Actb mRNA。
    7. 使用ΔΔCt方法分析结果(Pfaffl MW 2001)。首先将来自一式两份或三份样品的Ct值归一化为它们各自的内部管家转录物,在该实施例中为Actb mRNA,然后在加入放线菌素D之前归一化为它们各自的样品,其设置为1.结果表示为mRNA丰度相对于时间0.代表性实验显示在表1中 注意:在使用前验证引物扩增效率和特异性是至关重要的。为了检查引物扩增效率,使用跨越4-5个对数范围的cDNA的10倍连续稀释。将内部管家转录物归一化后的相对Ct值对线性回归曲线的对数值的浓度作图。当小于或等于0.1的斜率的任何绝对值在测定中与管家基因配对时,被认为通过效率测试。代表性实验如图1所示。

      表1.使用ΔΔCt方法计算相对转录丰度

      *ΔCt= Ct(Fos,column 2) - Ave Ct(Actb,column 4); **ΔΔCt=ΔCt(第5列)-AveΔCt在时间0(第6列)。在TNF刺激30分钟后加入放线菌素D(ActD)
    8. 然后使用GraphPad Prism软件基于非线性拟合单相指数衰减曲线计算转录物转换率,并且通常表示为每个实验的50%mRNA衰减的时间。一个代表性的实验如图2所示

代表数据



图1.实时PCR引物的验证制备在10,5,1,0.5,0.1和0.01ng的cDNA的系列稀释液作为模板,用于实时PCR分析,使用针对Fos的引物和Actb转录物。所示图中的X轴是cDNA的对数值;在Y轴上显示的ΔCt是在Fos 和 Actb mRNA之间的Ct值的差异。呈现的数据是来自一个代表性实验的重复的平均值±SD。绘制线性回归曲线,绝对斜率值≤0.1。在该实施例中,0.01ng时Fos转录物的Ct值不可检测,因此不包括在回归曲线的计算中。


图2.在存在和不存在TTP的情况下TNF刺激后Fos和Cxcl1转录物的稳定性。在血清饥饿的成纤维细胞系的TNF处理30分钟后加入放线菌素D以抑制转录。在指定的时间收获细胞用于总RNA提取,并且用实时RT-PCR测量mRNA水平。将转录物浓度相对于Actb转录物的浓度标准化,并且在加入放线菌素D之前以TNF处理的样品中的丰度分数表示。所示结果是来自一个代表性实验的重复样品的平均值。使用GraphPad软件(红色虚线,66KO细胞;蓝色虚线,67个WT细胞)基于非线性拟合单相指数衰减曲线计算转录物转换率,并且表达为每次50%mRNA衰减的时间实验(当50%mRNA衰减线的黑色虚线分别与每条曲线的红色和蓝色虚线交叉时的时间点)。

食谱

  1. 完成媒介
    DMEM
    10%FBS
    1%Pen-Strep
    2mM谷氨酰胺
  2. 血清饥饿培养基
    DMEM
    0.5%FBS
    1%Pen-Strep
    2mM谷氨酰胺

致谢

该方案改编自先前公开的研究,Lai等人(2006)和Horner等人(2009),并且如本文所述使用, et al。 (2015)。我们感谢博士。 Melissa Wells和Diana Cruz-Topete对协议的意见。这项研究由国立环境卫生科学研究所,国立卫生研究院的校内研究计划支持。

参考文献

  1. Horner,TJ,Lai,WS,Stumpo,DJ和Blackshear,PJ(2009)。 
  2. Lai,WS,Parker,JS,Grissom,SF,Stumpo,DJ和Blackshear,PJ(2006)。  通过在TTP缺陷成纤维细胞中稳定转录物的全面分析鉴定的三四脯氨酸(TTP)的新型mRNA靶标。 :9196-9208。
  3. Pfaffl,MW(2001)。  一个新的数学模型在实时RT-PCR中的相对定量。 Nucleic Acids Res 29(9):e45。
  4. Qiu,LQ,Lai,WS,Bradbury,A.,Zeldin,DC和Blackshear,PJ(2015)。  三四脯氨酸(TTP)协调调节原发性和继发性细胞对促炎症刺激的反应。 Leukoc Biol 97(4):723-736。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Qiu, L. Q., Lai, W. S., Stumpo, D. J. and Blackshear, P. J. (2016). Measurement of mRNA Decay in Mouse Embryonic Fibroblasts. Bio-protocol 6(13): e1858. DOI: 10.21769/BioProtoc.1858.
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