Single cell expression levels of MBNL1 and DMPKare highly variable and no correlation was found at single cell level
After RNA foci visualization, RNA was obtained from every single myoblast for transcript analysis. Even though we could detect by qPCR all the transcripts of interest in myoblast pools (DMPK ,MBNL1 , INSR and ATP2A1 ), detection rate at single cell level was very low for most transcripts and only the exon 7 exclusion MBNL1 splicing variant (normal variant) and DMPKexpression levels could be analyzed. No correlation at the single cell level was found between the number of foci and DMPK or theMBNL1 exon 7 exclusion isoform at single cell level (Table S1 ).
We found that the exon 7 exclusion MBNL1 variant was significantly (p = 0.01) more expressed in control than in patient’s myoblasts, whereas no differences were found in DMPK expression levels between controls and patients (p = 0.1) (Figure 4 ).DMPK expression levels significantly differed (p- < 0.001) among patients, but all patient myoblasts showed similar expression levels for the MBNL1 exon 7 exclusion isoform (p = 0.8). We found variability in the expression of DMPK andMBNL1 exon 7 exclusion isoform among the different control individuals (p < 0.001) (Figure 5 ).
To allow for a better understanding of DMPK expression differences between patients and to determine if transcript levels were derived from the expanded or the normal allele, we searched for a single nucleotide polymorphism (SNP) that allowed differentiating expanded from normal transcripts in single cells. When testing our patients for the presence of the informative SNP, described in exon 10 (Korneluk, 1993), we found all of them to be homozygous, and we could therefore not use this SNP for the intended purpose.