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. 2022;2434:207–215. doi: 10.1007/978-1-0716-2010-6_13

Rapid Determination of MBNL1 Protein Levels by Quantitative Dot Blot for the Evaluation of Antisense Oligonucleotides in Myotonic Dystrophy Myoblasts.

Nerea Moreno, Irene González-Martínez, Rubén Artero, Estefanía Cerro-Herreros
PMCID: PMC9703865  PMID: 35213019

Abstract

Western blot assays are not adequate for high-throughput screening of protein expression because it is an expensive and time-consuming technique. Here we demonstrate that quantitative dot blots in plate format are a better option to determine the absolute contents of a given protein in less than 48 h. The method was optimized for the detection of the Muscleblind-like 1 protein in patient-derived myoblasts treated with a collection of more than 100 experimental oligonucleotides.

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References

  1. Ashizawa T, Gagnon C, Groh WJ, Gutmann L, Johnson NE, Meola G, Moxley R III, Pandya S, Rogers MT, Simpson E, Angeard N, Bassez G, Berggren KN, Bhakta D, Bozzali M, Broderick A, Byrne JLB, Campbell C, Cup E, Day JW, De Mattia E, Duboc D, Duong T, Eichinger K, Ekstrom AB, van Engelen B, Esparis B, Eymard B, Ferschl M, Gadalla SM, Gallais B, Goodglick T, Heatwole C, Hilbert J, Holland V, Kierkegaard M, Koopman WJ, Lane K, Maas D, Mankodi A, Mathews KD, Monckton DG, Moser D, Nazarian S, Nguyen L, Nopoulos P, Petty R, Phetteplace J, Puymirat J, Raman S, Richer L, Roma E, Sampson J, Sansone V, Schoser B, Sterling L, Statland J, Subramony SH, Tian C, Trujillo C, Tomaselli G, Turner C, Venance S, Verma A, White M, Winblad S (2018) Consensus-based care recommendations for adults with myotonic dystrophy type 1. Neurol Clin Pract 8(6):507–520. https://doi.org/10.1212/CPJ.0000000000000531 doi: 10.1212/CPJ.0000000000000531. [DOI] [PMC free article] [PubMed]
  2. Jiang H, Mankodi A, Swanson MS, Moxley RT, Thornton CA (2004) Myotonic dystrophy type 1 is associated with nuclear foci of mutant RNA, sequestration of muscleblind proteins and deregulated alternative splicing in neurons. Hum Mol Genet 13(24):3079–3088. https://doi.org/10.1093/hmg/ddh327 doi: 10.1093/hmg/ddh327. [DOI] [PubMed]
  3. Chamberlain CM, Ranum LP (2012) Mouse model of muscleblind-like 1 overexpression: skeletal muscle effects and therapeutic promise. Hum Mol Genet 21(21):4645–4654. https://doi.org/10.1093/hmg/dds306 doi: 10.1093/hmg/dds306. [DOI] [PMC free article] [PubMed]
  4. Landfeldt E, Nikolenko N, Jimenez-Moreno C, Cumming S, Monckton DG, Gorman G, Turner C, Lochmuller H (2019) Disease burden of myotonic dystrophy type 1. J Neurol 266(4):998–1006. https://doi.org/10.1007/s00415-019-09228-w doi: 10.1007/s00415-019-09228-w. [DOI] [PMC free article] [PubMed]
  5. Mathieu J, Allard P, Potvin L, Prevost C, Begin P (1999) A 10-year study of mortality in a cohort of patients with myotonic dystrophy. Neurology 52(8):1658–1662 doi: 10.1212/wnl.52.8.1658. [DOI] [PubMed]
  6. Arandel L, Polay Espinoza M, Matloka M, Bazinet A, De Dea DD, Naouar N, Rau F, Jollet A, Edom-Vovard F, Mamchaoui K, Tarnopolsky M, Puymirat J, Battail C, Boland A, Deleuze JF, Mouly V, Klein AF, Furling D (2017) Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds. Dis Model Mech 10(4):487–497. https://doi.org/10.1242/dmm.027367 doi: 10.1242/dmm.027367. [DOI] [PMC free article] [PubMed]
  7. Cerro-Herreros E, Sabater-Arcis M, Fernandez-Costa JM, Moreno N, Perez-Alonso M, Llamusi B, Artero R (2018) miR-23b and miR-218 silencing increase Muscleblind-like expression and alleviate myotonic dystrophy phenotypes in mammalian models. Nat Commun 9(1):2482. https://doi.org/10.1038/s41467-018-04892-4 doi: 10.1038/s41467-018-04892-4. [DOI] [PMC free article] [PubMed]
  8. Crooke ST, Witztum JL, Bennett CF, Baker BF (2019) RNA-targeted therapeutics. Cell Metab 29(2):501. https://doi.org/10.1016/j.cmet.2019.01.001 doi: 10.1016/j.cmet.2019.01.001. [DOI] [PubMed]
  9. Smith CIE, Zain R (2019) Therapeutic oligonucleotides: state of the art. Annu Rev Pharmacol Toxicol 59:605–630. https://doi.org/10.1146/annurev-pharmtox-010818-021050 doi: 10.1146/annurev-pharmtox-010818-021050. [DOI] [PubMed]
  10. Tian G, Tang F, Yang C, Zhang W, Bergquist J, Wang B, Mi J, Zhang J (2017) Quantitative dot blot analysis (QDB), a versatile high throughput immunoblot method. Oncotarget 8(35):58553–58562. https://doi.org/10.18632/oncotarget.17236 doi: 10.18632/oncotarget.17236. [DOI] [PMC free article] [PubMed]
  11. Qi X, Zhang Y, Zhang Y, Ni T, Zhang W, Yang C, Mi J, Zhang J, Tian G (2018) High throughput, absolute determination of the content of a selected protein at tissue levels using quantitative dot blot analysis (QDB). J Vis Exp (138):56885. https://doi.org/10.3791/56885 doi: 10.3791/56885. [DOI] [PMC free article] [PubMed]

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