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,Estefanía Cerro-Herreros,Rubén Artero

doi:10.1007/978-1-0716-2010-6_13

Nerea Moreno, Irene González-Martínez + Show 2 more

Open Access

https://doi.org/10.1007/978-1-0716-2010-6_13

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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 48h. 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.

Highlights

Myotonic dystrophy type 1 (DM1) is a degenerative genetic disease that is classified as rare because it affects less than 1 in 2000 people (1/3000 to 1/8000; [1])
DM1 originates from an expansion of the CTG trinucleotide repeat in the 30-untranslated region (UTR) of the DMPK gene that, upon transcription, forms CUG hairpins that behave as toxic RNAs
One example is the need to quickly evaluate the levels of MBNL1 protein in patientderived myoblasts [6] treated with hundreds of oligonucleotide variants to block repressive miRNAs miR-23b and miR-218, as a means to boost endogenous levels and compensate sequestration by CUG expansions in mutant DMPK [7]

Summary

Introduction

Myotonic dystrophy type 1 (DM1) is a degenerative genetic disease that is classified as rare because it affects less than 1 in 2000 people (1/3000 to 1/8000; [1]). One example is the need to quickly evaluate the levels of MBNL1 protein in patientderived myoblasts [6] treated with hundreds of oligonucleotide variants to block repressive miRNAs miR-23b and miR-218, as a means to boost endogenous levels and compensate sequestration by CUG expansions in mutant DMPK [7]. To this end, we have generated a diversity (>100) of highly modified antisense oligonucleotides (AONs) to block miR-23b and miR-218. Rapid Determination of MBNL1 Protein Levels by Quantitative Dot Blot for. . . 209 Fig. 1 Illustration of the entire QDB process for evaluation of antisense oligonucleotides in DM1 myoblast

QDB Assay

Transfer buffer

Sample Preparation

Quantification

Data Analysis