Fluorescence in situ hybridization (FISH)
To visualize the RNA foci in single myoblasts before RNA analysis, a
quick FISH staining method was developed. The aim of this method was to
preserve RNA integrity as much as possible. In brief, myoblast pellets
were resuspended in formamide and incubated for 20 min at 37ºC.
Myoblasts were then incubated with the hybridization buffer containing
AT0488 labelled (CAG)10 probe 0.01 uM and 30% formamide
in 2XSSC buffer for 20 min at 37ºC. Myoblasts were then washed with 30%
formamide 2x SSC, 1x SSC and 1x PBS and incubated for 5 min with 5 ul of
DAPI mounting solution. Finally, myoblasts were resuspended with 500 µl
of 1x PBS.