Skeletal muscle ribosome analysis: a comparison of common assay methods and utilization of a novel RiboAb antibody cocktail
Cellular and tissue total RNA concentrations have been widely reported to represent ribosome content, a metric that reflects the trophic state of skeletal muscle. Although various assays are used to assess total RNA concentrations, there is a need to homologize the various quantification approaches. Thus, we analyzed C2C12 myotubes and mouse skeletal muscle to determine if total RNA concentrations provided through UV-Vis spectroscopy (UV), fluorometry only (Fluor), and fluorometry-based microfluidic chip electrophoresis (MFGE) were representative of cellular and muscle tissue rRNA concentrations (i.e., MFGE 18S+28S rRNA, criterion metric of ribosome content). We also sought to determine whether a novel ribosomal protein antibody cocktail (termed RiboAb) corresponded with 18S+28S rRNA concentrations. Compared to non-treated C2C12 myotubes, 24-hour insulin-like growth factor-1 (IGF-1) treatments increased 18S+28S rRNA concentrations ([~]2.0-fold; p<0.001) and total RNA concentrations based on UV ([~]1.9-fold; p<0.001), Fluor ([~]2.3 fold; p=0.001), and MFGE ([~]2.1-fold, p<0.001). In C57BL/6 mice, 10 days of mechanical overload (MOV) via synergist ablation elevated plantaris 18S+28S rRNA concentrations ([~]1.7-fold; p=0.017) and total RNA concentrations according to UV ([~]1.5-fold; p=0.033), Fluor ([~]1.6-fold; p=0.001), and MFGE ([~]1.8-fold, p=0.017). In both experiments, total RNA concentration data yielded from all three techniques exhibited significant positive correlations to 18S+28S rRNA concentrations. Ribosome pelleting experiments indicated that the proteins assayed with the RiboAb cocktail (rps3/6 and rpl5/11) were exclusively associated with the ribosome pellet. Additionally, C2C12 myotube and mouse plantaris RiboAb levels were higher with IGF-1 treatments and MOV, respectively, relative to controls (1.3-fold and 1.7-fold, respectively, p<0.017), and values correlated with rRNA concentrations (r=0.637 and r=0.853, respectively, p<0.005). These data confirm that total RNA concentrations yielded from the UV, Fluor, MFGE techniques are valid surrogates of cell/tissue ribosome content. We also propose that the RiboAb cocktail may serve as a surrogate for changes in ribosome content in these models, although future research is needed to examine the feasibility of the RiboAb cocktail in humans as well as utility with other applications (e.g., immunohistochemistry and/or tissue fractionation experiments).