Myoblast isolation and fluorescence microscope visualization
For this purpose, two different approaches were tested: 1) single cell C1 Fluidigm platform (Fluidigm, South San Francisco, CA) and 2) sorting cytometry. The first approach, single cell C1 Fluidigm platform, was especially suitable for our purpose because isolated cells can be visualized through microscopy and after, RNA extraction and cDNA synthesis can be made in an automated system that works with thermal cycling and pneumatic controls, minimizing the variation between conditions and expending only small amounts of reagents in every plate. To make the analysis with the C1 Fluidigm single cell platform, myoblasts were resuspended in C1 suspension reagent and loaded into the C1 IFC chip, that contains 96 single cell capture sites. After this process, myoblasts were captured in single and isolated chambers in the IFC and then observed by fluorescent microscopy at 40X to count foci number. Unfortunately, we failed to detect foci fluorescent signal (see results section), so we decided to do this process through our second approach, sorting cytometry. For this second approach, cells were sorted using FACSAria II flow cytometer (BD Biosciences, San Jose, CA) into 96-Well Optical-Bottom Plates. For each participant, we sorted a total of 120 FISH stained myoblasts distributed in two plates. The cytometer could not detect the fluorescent signal of the FISH staining (probably because RNA foci fluorescence signal is small and because of their nuclear localization). Therefore, sorting was only used to isolate individual myoblasts. RNA foci signal was observed by fluorescent microscopy. In order to spend the least time possible, since these same cells were later used for RNA analysis, and because there were two plates to analyze per participant, two independent observers simultaneously visualized one plate each, in similar fluorescence microscopes. They had the same criteria for annotating RNA foci number in every single myoblast by naked eye visualization.