Single cell analysis of RNA foci shows a heterogeneous behavior between and within patients´ myoblasts, with several molecular alterations linked to muscle function
For these experiments, we developed a fast protocol of RNA foci staining, in order to preserve RNA for posterior analysis. Two approaches were used to perform RNA foci single cell quantification: single cell Fluidigm C1 platform; and sorting cytometry.
In the first approach, previously stained, single myoblasts were captured into the C1 IFC chip and were visible by bright field microscopy (Figure 1A ). 4′,6-diamidino-2-phenylindole (DAPI) fluorescent signal was also visible in captured myoblasts. However, we failed to detect RNA foci fluorescent signal (Figure 1B ), but the latter could be detected in the same cells and with the same staining when using standard immunocytochemistry (Figure 1C ). We found incompatibility of the focal distance of the microscope with the thickness of the chip, which was much higher and made it impossible to focus on such small particles as the RNA foci.
Due to the problems found in the first approach, we started a second approach based on sorting cytometry. By this technique, we were able to isolate and count foci from 39 to 72 single myoblasts (depending on the patient) of 120 myoblasts that were sorted for every patient (Table S1 ). The wells from the 96-well plates used to sort the myoblasts were analyzed with a fluorescent microscope. The number of RNA foci per myoblast was manually annotated from right to left and top to bottom in the plates.
We found evidence of heterogeneity in myoblast foci number both between and within patients (Figure 2 , Table 1 ). The percentage of DM1 myoblasts with RNA foci was high in three of the cell lines (>80%). However, the distribution of RNA foci per myoblast was very different in every patient (Figure 3 ). In all myoblast cell lines we could find some myoblasts without foci, indicating a dynamic process of foci accumulation, that reached a maximum of 7 RNA foci in some myoblasts. However, when looking at all the DM1 myoblasts analyzed in this study, we concluded that most of them were carrying between 0 to 3 RNA foci (20% had 0 RNA foci, 21% 1 RNA foci, 24% 2 RNA foci, 22% 3 RNA foci, 10% 4 RNA foci, and 2% 5-7 RNA foci). No foci was observed in the myoblasts of healthy controls. Although no correlation with CTG repeat number was found, P5 (who was the carrier of the shortest CTG expansion in myoblasts) was showing the highest number of myoblasts without RNA foci. Regarding muscle function and disability, P4 (the most severely affected patient) had more myoblasts carrying numerous foci than the rest of the patients and less myoblasts without RNA foci. We found a correlation between 6MWD and the mean number of foci per cell (p=0.017) and foci median (p=0.005).