Overall, JetOPTIMUS® led to the highest percentage of transfected myoblasts, regardless of plasmid size, with the smallest amount of reagent used. Finally, Avalanche® and JetOPTIMUS® had a limited effect on cell viability and cell growth. Moreover, they were compatible with cell differentiation and the generation of GFP-positive myotubes. Of note, none of these reagents gave GFP-positive myotubes when performing the transfection after inducing differentiation. Hence, considering their limited effect on cell dynamics and their transfection efficiency at limited cost (volume used/cost of the reagent), JetOPTIMUS®, and to a lesser extent Avalanche®, constitute the best option to transfect C2C12 myoblasts. Cytoplasmic fluorescent puncta corresponding to siRNA accumulation were detected in 79.2% and 98.3% of cells transfected with Lipofectamine®RNAiMAX and INTERFERin®, respectively. In parallel, the proportion of transfected cells with Viafect™ and JetOPTIMUS® yielded more than 50% cells (Fig. 5C). In contrast, very few siRNA-positive cells were observed using Fugene® HD, Lipofectamine®3000, or Avalanche®.

C2C12 cells are widely used in the muscle field, as they differentiate easily into myotubes and show limited constraints to culture as compared to primary myoblasts. Both C2C12 and primary myoblasts are hard to transfect, which affects downstream experiments. More than 95% of the reports published since 2015 with C2C12 cells have used one gold standard transfectant (i.e., Lipofectamine®), although several studies have suggested less than 30% efficiency of this reagent. In parallel, the capacity of other commercial reagents to transfect muscle cells remains largely unknown. Here, we compared transfection efficiency of five commercial reagents (Lipofectamine® 3000, Viafect™, Fugene® HD, C2C12 Cell Avalanche®, and JetOPTIMUS®) in C2C12 cells. By optimizing DNA:transfectant ratios and cell density, all reagents reached more than 60% transfection efficiency with limited effects on cell growth and viability. GFP-positive myotubes were efficiently generated in cultures transfected with Lipofectamine® 3000, Fugene® HD, C2C12 Cell Avalanche®, and JetOPTIMUS®. Notably, in conditions optimized for DNA transfer in C2C12 cells, these reagents showed low efficiency to transfer siRNA and higher toxicity for primary muscle cells. In conclusion, we reported yet uncharacterized transfection reagents that can serve as a suitable low-cost alternative to the current gold standard in C2C12 cells.