Analysis of N⁶-methyladenosine RNA Modification Levels by Dot Blotting

Materials and Reagents

1. 100 mm culture dish (Corning, Falcon, catalog number: 353003)

2. Pipette tips (Axygen, catalog numbers: T-300, T-200Y, T-1000B)

3. 1.5 mL RNase/DNase-free microcentrifuge tube (Axygen, catalog numbers: MCT-50-C)

4. Cell scraper (any brand)

5. TRIzol reagent (Invitrogen, catalog number:15596026)

6. Chloroform (General-Reagent, catalog number: G75915B)

7. Isopropanol (Sangon Biotech, catalog number: A507048)

8. Ethanol absolute (Sangon Biotech, catalog number: A500737)

9. RNase-free water

10. GenElute mRNA miniprep kit (Sigma, catalog number: DMN10)

11. Amersham^TM Hybond^TM-N+ membrane (GE Healthcare, catalog number: RPN203B )

12. N⁶-methyladenosine/m⁶A rabbit mAb (ABclonal, catalog number: A19841)

13. HRP goat anti-rabbit IgG (H+L) (ABclonal, catalog number: AS014)

14. ECL western blotting substrate (Thermo Fisher Scientific, catalog number: 32106)

15. Saran wrap (any brand)

16. Tween-20 (Sigma-Aldrich, catalog number: P1379)

17. Non-fat milk (any brand)

18. Na₂HPO₄ (Sangon Biotech, catalog number: A501727)

19. KH₂PO₄ (Sangon Biotech, catalog number: A100781)

20. KCl (Sangon Biotech, catalog number: A100395)

21. NaCl (Sangon Biotech, catalog number: A501218)

22. 10× phosphate-buffered saline (PBS) (see Recipes)

23. 1× phosphate-buffered saline (PBS) (see Recipes)

24. 1× phosphate-buffered saline/0.1% Tween-20 (PBST) (see Recipes)

25. Blocking buffer (see Recipes)

26. Antibody dilution buffer (see Recipes)

27. Methylene blue dye buffer (Solarbio, catalog number: G1301)

Equipment

1. Refrigerated centrifuge (Eppendorf, model: 5424 R)

2. NanoDrop (Thermo Fisher Scientific, model: ND-1000 spectrophotometer)

3. UV cross-linker or UV torch with 254 nm wavelength UV (UVP, model: CL1000)

4. Chemiluminescent imaging system (Tanon, model: Tanon 5200)

5. Camera (any brand)

Software

1. GraphPad Prism (GraphPad Software)

2. ImageJ (https://imagej.nih.gov/ij)

Procedure

1. Isolation of total RNA

   1. For mammalian cells we recommend using approximately 1 × 10⁷ cells for mRNA purification.

   2. Remove the medium and rinse the cells twice with 1–2 mL of ice-cold PBS.

   3. Remove PBS and lyse the cells directly in the culture dish by adding 1 mL of TRIzol reagent per 100 mm culture dish and scraping with a cell scraper.

   4. Pass the cell lysate through a pipette several times. Then, transfer to 1.5 mL RNase-free microcentrifuge tubes. Vortex thoroughly.

   5. Incubate the homogenized samples for 5 min at room temperature.

   6. Add 0.2 mL of chloroform, vortex for 30 s, and incubate for 2–3 min at room temperature.

   7. Centrifuge at 12,000 × g and 4 °C for 15 min.

   8. Carefully transfer the upper, aqueous phase to a fresh tube without disturbing the interface.

   9. Add 0.5 mL of isopropanol to the aqueous phase and mix well. Incubate the samples for 10 min at room temperature.

   10. Centrifuge at 12,000 × g and 4 °C for 10 min.

   11. Decant the supernatant and completely remove any traces of liquid by aspiration.

   12. Wash the pellet with 1 mL of ice-cold 75% ethanol.

   13. Centrifuge at 12,000 × g and 4 °C for 15 min.

   14. Remove all traces of ethanol. Air-dry or vacuum-dry the RNA pellet for 5–10 min.

   15. Resuspend the pellet in 10–30 μL of RNase-free water.

       
       

2. mRNA purification

   1. Isolate mRNA from total RNA using the GenElute mRNA miniprep kit following the manufacturer’s instructions (other brands can also be used for the purification of mRNA).

   2. Mix the oligo (dT) beads thoroughly by vortexing and inverting until resuspended and homogenous.

   3. Add the resuspended oligo (dT) beads to the total RNA, cap the tube, and mix thoroughly by vortexing.

   4. Incubate the mixture at room temperature for 10 min. No mixing or rocking is necessary.

   5. Resuspend the pellet in 350 μL of wash solution by vortexing.

   6. Transfer the suspension into a GenElute spin filter–collection tube assembly by pipetting. Spin for 1–2 min at maximum speed.

   7. Remove the spin filter, discard the flow through liquid, then place the spin filter back into the same collection tube.

   8. Pipette 350 μL of low salt wash solution into the column. Spin for 1–2 min at maximum speed.

   9. Remove the spin filter, discard the flow through liquid, then place the spin filter back into the same collection tube.

   10. Pipette another 350 μL of low salt wash solution into the column. Spin for 1–2 min at maximum speed.

   11. Transfer the spin filter into a fresh collection tube. Pipette 10–20 μL of preheated (65 °C) elution solution onto the spin filter, ensuring that it comes into contact with the bead–mRNA complex. Incubate for 2–5 minutes at 65 °C. Spin for 1–2 min at maximum speed. Save the flow through liquid; it contains most of the purified mRNA.

   12. Determine the concentration of purified mRNA with NanoDrop.

   13. Dilute different concentrations of mRNA to 50 ng/μL.

       
       

3. Dot blotting

   1. The isolated mRNA is first denatured by heating at 95 °C for 5 min to disrupt the secondary structure.

   2. Chill on ice for 5 min immediately after denaturation to prevent re-formation of the mRNA secondary structure.

   3. Have the nitrocellulose membrane ready.

   4. Using a narrow-mouth pipette tip, drop 2 μL of mRNA sample directly onto the Hybond-N+ membrane at the center of the grid (Figure 2).

      
      

      
      Figure 2. Dot sample example

      
      

   5. Air dry at room temperature for 5 min.

   6. Crosslink spotted RNA to the membrane using the UV cross linker (irradiate under ultraviolet lamp with 254 nm wavelength for 5 min).

   7. Incubate the membrane with blocking buffer for 1 h at room temperature.

   8. After blocking, incubate the membrane with anti-m6A antibody (1:1,000 dilution) in 5 mL of antibody dilution buffer overnight at 4 °C with gentle shaking.

   9. Wash three times with PBST (3 × 5 min).

   10. Incubate the membrane with HRP goat anti-rabbit IgG (1:10,000 dilution) in 5 mL of antibody dilution buffer for 1 h at room temperature with gentle shaking.

   11. Wash three times with PBST (3 × 5 min).

   12. Incubate with ECL substrate for 1 min (according to the manufacturer’s instructions, 0.125 mL ECL solution per cm² of the membrane is recommended), cover with Saran wrap aiming to remove the excess solution from the surface, and expose the film in the darkroom (Figure 3B). Try different exposure times to get clear results.

   13. After exposure, transfer the membrane to a solution containing 10 mL of 0.02% methylene blue dye buffer, gently shake the membrane at room temperature, and incubate for 30 min.

   14. Lastly, clean the membrane with dH₂O until the background is clean (Figure 3C).

       
       

   
   Figure 3. Representative data of m⁶A level detected by dot blot. (A) Western blotting assay of METTL3 in the METTL3-KD stable cell lines in MCF-7. (B) Dot blot to detect the m⁶A level of mRNA isolated from total RNA of METTL3-KD MCF-7 cells. (C) Methylene blue (MB) staining served as a loading control. (D) Measurement and normalization of m⁶A levels in ImageJ.

Data analysis

1. Perform densitometric analysis using ImageJ (result calculation method: m⁶A grayscale value/MB grayscale value). Use a minimum of three biological replicates to perform statistical analysis of the m⁶A levels between samples (Figure 3D).

2. For statistical analysis, use GraphPad Prism software.

Notes

1. This protocol can be modified for individual species of RNA, such as messenger RNA, tRNA, rRNA, or microRNA, by employing isolation techniques for the enrichment of the specific RNAs to be analyzed.

2. For the preparation of wash buffer, blocking buffer, and antibody dilution buffer, the use of nuclease-free water is not necessary.

Recipes

1. 10× phosphate-buffered saline (PBS) (1,000 mL)

   1. Weigh 14.4 g of Na₂HPO₄, 2.4 g of KH₂PO₄, 2.0 g of KCl, and 80.0 g of NaCl

   2. Add 800 mL of sterile water

   3. Stir until dissolved

   4. Bring the final volume to 1,000 mL using sterile water

      
      

2. 1× phosphate-buffered saline (PBS) (500 mL)

   50 mL of 10× PBS

   Bring final volume to 500 mL using sterile water

   
   

3. 1× phosphate-buffered saline/0.1% Tween-20 (PBST) (500 mL)

   50 mL of 1× PBS

   1 mL of 100% Tween-20

   Bring final volume to 500 mL using sterile water

   
   

4. Blocking buffer and antibody dilution buffer

   50 mL of PBST

   5 g non-fat milk

Acknowledgments

This work was supported by a grant from the National Natural Science Foundation of China (32171407) and Priority Academic Program Development of Jiangsu Higher Education Institutions. The protocol presented here was adapted from those in Terenghi (1998) and Jia et al. (2011).

Competing interests

The author declares there are no competing interests.

References

1. Arguello, A. E., DeLiberto, A. N. and Kleiner, R. E. (2017). RNA Chemical Proteomics Reveals the N⁶-Methyladenosine (m⁶A)-Regulated Protein-RNA Interactome. J Am Chem Soc 139(48): 17249-17252.
2. Batista, P. J., Molinie, B., Wang, J., Qu, K., Zhang, J., Li, L., Bouley, D. M., Lujan, E., Haddad, B., Daneshvar, K., et al. (2014). m⁶A RNA modification controls cell fate transition in mammalian embryonic stem cells. Cell Stem Cell 15(6): 707-719.
3. Chen, W., Feng, P., Ding, H. and Lin, H. (2016). Identifying N⁶-methyladenosine sites in the Arabidopsis thaliana transcriptome. Mol Genet Genomics 291(6): 2225-2229.
4. Csepany, T., Lin, A., Baldick, C. J., Jr. and Beemon, K. (1990). Sequence specificity of mRNA N⁶-adenosine methyltransferase. J Biol Chem 265(33): 20117-20122.
5. Engel, M., Eggert, C., Kaplick, P. M., Eder, M., Roh, S., Tietze, L., Namendorf, C., Arloth, J., Weber, P., Rex-Haffner, M., et al. (2018). The Role of m⁶A/m-RNA Methylation in Stress Response Regulation. Neuron 99(2): 389-403 e389.
6. Harcourt, E. M., Kietrys, A. M. and Kool, E. T. (2017). Chemical and structural effects of base modifications in messenger RNA. Nature 541(7637): 339-346.
7. Imanishi, M., Tsuji, S., Suda, A. and Futaki, S. (2017). Detection of N⁶-methyladenosine based on the methyl-sensitivity of MazF RNA endonuclease. Chem Commun (Camb) 53(96): 12930-12933.
8. Jia, G., Fu, Y. and He, C. (2013). Reversible RNA adenosine methylation in biological regulation. Trends Genet 29(2): 108-115.
9. Jia, G., Fu, Y., Zhao, X., Dai, Q., Zheng, G., Yang, Y., Yi, C., Lindahl, T., Pan, T., Yang, Y. G., et al. (2011). N⁶-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol 7(12): 885-887.
10. Li, E., Xia, M., Du, Y., Long, K., Ji, F., Pan, F., He, L., Hu, Z. and Guo, Z. (2022). METTL3 promotes homologous recombination repair and modulates chemotherapeutic response in breast cancer by regulating the EGF/RAD51 axis. Elife 11: e75231.
11. Li, Z., Weng, H., Su, R., Weng, X., Zuo, Z., Li, C., Huang, H., Nachtergaele, S., Dong, L., Hu, C., et al. (2017). FTO Plays an Oncogenic Role in Acute Myeloid Leukemia as a N⁶-Methyladenosine RNA Demethylase. Cancer Cell 31(1): 127-141.
12. Linder, B., Grozhik, A. V., Olarerin-George, A. O., Meydan, C., Mason, C. E. and Jaffrey, S. R. (2015). Single-nucleotide-resolution mapping of m⁶A and m⁶Am throughout the transcriptome. Nat Methods 12(8): 767-772.
13. Liu, H., Wang, H., Wei, Z., Zhang, S., Hua, G., Zhang, S. W., Zhang, L., Gao, S. J., Meng, J., Chen, X., et al. (2018). MeT-DB V2.0: elucidating context-specific functions of N⁶-methyl-adenosine methyltranscriptome. Nucleic Acids Res 46(D1): D281-D287.
14. Mishima, E., Jinno, D., Akiyama, Y., Itoh, K., Nankumo, S., Shima, H., Kikuchi, K., Takeuchi, Y., Elkordy, A., Suzuki, T., et al. (2015). Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides. PLoS One 10(11): e0143756.
15. Nachtergaele, S. and He, C. (2018). Chemical Modifications in the Life of an mRNA Transcript. Annu Rev Genet 52: 349-372.
16. Niu, Y., Zhao, X., Wu, Y. S., Li, M. M., Wang, X. J. and Yang, Y. G. (2013). N⁶-methyl-adenosine (m⁶A) in RNA: an old modification with a novel epigenetic function. Genomics Proteomics Bioinformatics 11(1): 8-17.
17. Rana, A. P. and Tuck, M. T. (1990). Analysis and in vitro localization of internal methylated adenine residues in dihydrofolate reductase mRNA. Nucleic Acids Res 18(16): 4803-4808.
18. Sun, T., Wu, R. and Ming, L. (2019). The role of m⁶A RNA methylation in cancer. Biomed Pharmacother 112: 108613.
19. Terenghi, G. (1998). Detecting mRNA in tissue sections with digoxigenin-labeled probes. Methods Mol Biol 86: 137-142.
20. Wang, Y., Li, Y., Yue, M., Wang, J., Kumar, S., Wechsler-Reya, R. J., Zhang, Z., Ogawa, Y., Kellis, M., Duester, G., et al. (2018). N⁶-methyladenosine RNA modification regulates embryonic neural stem cell self-renewal through histone modifications. Nat Neurosci 21(2): 195-206.
21. Wiedmer, L., Eberle, S. A., Bedi, R. K., Sledz, P. and Caflisch, A. (2019). A Reader-Based Assay for m⁶A Writers and Erasers. Anal Chem 91(4): 3078-3084.
22. Winkler, R., Gillis, E., Lasman, L., Safra, M., Geula, S., Soyris, C., Nachshon, A., Tai-Schmiedel, J., Friedman, N., Le-Trilling, V. T. K., et al. (2019). m⁶A modification controls the innate immune response to infection by targeting type I interferons. Nat Immunol 20(2): 173-182.
23. Xiang, Y., Laurent, B., Hsu, C. H., Nachtergaele, S., Lu, Z., Sheng, W., Xu, C., Chen, H., Ouyang, J., Wang, S., et al. (2017). RNA m⁶A methylation regulates the ultraviolet-induced DNA damage response. Nature 543(7646): 573-576.
24. Yin, H., Wang, H., Jiang, W., Zhou, Y. and Ai, S. (2017). Electrochemical immunosensor for N⁶-methyladenosine detection in human cell lines based on biotin-streptavidin system and silver-SiO₂ signal amplification. Biosens Bioelectron 90: 494-500.
25. Zhou, J., Wan, J., Gao, X., Zhang, X., Jaffrey, S. R. and Qian, S. B. (2015). Dynamic m⁶A mRNA methylation directs translational control of heat shock response. Nature 526(7574): 591-594.