RNA isolation, cDNA synthesis, preamplification and quantitative
PCR (qPCR)
RNA from single myoblasts was isolated using GenElute™ Single Cell RNA
Purification Kit (Sigma-Aldrich, San Luis, MO) and cDNA was synthesized
using TruScript First Strand cDNA Synthesis Kit (Norgen Biotek Corp.,
Thorold, ON, Canada), following the manufacturer’s protocol in both.
Transcripts of interest were pre-amplified with gene specific Taqman
assays using TaqMan™ PreAmp Master Mix (ThermoFisher, Waltham, MA). This
pre-amplification step is used in most single cell studies. Transcripts
of interest included DMPK , MBNL1 exon 7 inclusion
(aberrant) and exclusion isoforms, ATP2A1 exon 22 inclusion and
exclusion (aberrant) isoforms and INSR exon 11 inclusion and
exclusion (aberrant) isoforms. The design of MBNL1 , INSRand ATP2A1 Taqman assays was done with the Assay Design Tool
(Thermofisher), while for DMPK we used a previously described
Taqman assay (Pandey et al., 2015). We analyzed the expression ofDMPK and INSR , ATP2A1 and MBNL1 splicing
variants by qPCR using Taqman Fast Advanced Master Mix (ThermoFisher)
and the same Taqman assays as in pre-amplification (Thermofisher). No
endogenous transcript was used for normalization because in single cell
analysis housekeeping genes can be misleading as their expression varies
from cell to cell (Ståhlberg & Kubista, 2018). Data analysis was
performed as previously described (Ståhlberg et al., 2013).