mRNA Stability Assay Using Transcription Inhibition by Actinomycin D in Mouse 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: Heracell^TM 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 Biosystems^TM, 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.
   

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.