Journal: Wellcome Open Research

Article Title: Single-transcript multiplex in situ hybridisation reveals unique patterns of dystrophin isoform expression in the developing mammalian embryo

doi: 10.12688/wellcomeopenres.15762.2

Figure Lengend Snippet: RNAscope probes to mouse dystrophin 5’ and 3’ regions recognise canine dystrophin in both healthy (WT) and dystrophic (deltaE50-MD) vastus lateralis muscle from 3-month-old ( A ) and 15-month-old ( C ) male animals. Nuclei (likely individual cells) associated with many 3’ probe foci are found within dystrophic muscle (arrowheads). Note dystrophin expression in blood vessel smooth muscle ( C , deltaE50-MD, lower panels). Apparent size distributions of probe foci ( B and D , upper panels) and subcellular localisations (lower panels) reveals 5’ foci fall into two populations: one small (~1.5 μm 2 ) and predominately sarcoplasmic; one large (>10 μm 2 ) and exclusively nuclear. 3’ foci are uniformly small (both sarcoplasmic and nuclear). DeltaE50-MD muscle shows no reduction in counts of sarcoplasmic 5’/3’ foci: instead nuclear counts of 3’ foci (commensurate with dp71 expression) tend to be higher. ( E ) qPCR for 5’ (exons 1-2) and 3’ (exons 62-64) regions of dp427m and for dp71 in 3- and 15-month-old WT and deltaE50-MD vastus lateralis muscles, confirms no significant dystrophy-associated reduction in dp427 transcriptional initiation and a modest (~2-fold) reduction in mature transcript number (P=0.006). A concomitant ~2-fold increase in dp71 expression (P=0.0005) is observed. All expression values are arbitrary units, normalised to HPRT1, RPL13a and SDHA (see methods) and adjusted relative to exon 1-2 values for comparative purposes. RNAscope ISH N=2-3 per genotype; qPCR N=6-7 per genotype, repeated measures 2-way ANOVA with Sidak’s multiple comparisons test (WT vs deltaE50-MD). Full-size figure can be found in the Underlying data .

Article Snippet: Statistical analysis of qPCR data ( vastus lateralis only) was performed with repeated measures 2-way ANOVA with Sidak’s multiple comparisons test (GraphPad Prism 8), with significance set at P<0.05.

Techniques: RNAscope, Expressing, Muscles