Skeletal muscle ribosome analysis: A comparison of common assay methods and utilization of a novel RiboAb antibody cocktail.

Abstract

While total RNA concentrations putatively represent ribosome content, there is a need to homologize various quantification approaches. Thus, total RNA concentrations ([RNA]) provided through UV-Vis spectroscopy (UV), fluorometry-only (Fluor), and fluorometry-based microfluidic chip electrophoresis (MFGE) were examined in C2C12 myotubes and mouse skeletal muscle to determine if values aligned with [18S + 28S rRNA] (i.e., criterion ribosome metric). A novel antibody cocktail (termed RiboAb) was also tested and compared to [18S + 28S rRNA] in these models. In myotubes, 24-h IGF-1 treatments increased [18S + 28S rRNA] (~2.0-fold) and [RNA] based on UV (~1.9-fold), Fluor (~2.3 fold), and MFGE (~2.1-fold). In C57BL/6 mice, 10 days of mechanical overload (MOV) elevated plantaris [18S + 28S rRNA] (~1.7-fold) and [RNA] according to UV (~1.5-fold), Fluor (~1.6-fold), and MFGE (~1.8-fold). Myotube and mouse plantaris RiboAb levels were significantly higher with IGF-1 treatments and MOV, respectively, versus controls (1.3-fold and 1.7-fold, respectively), and values correlated with [18S + 28S rRNA] (r = 0.637 and r = 0.853, respectively, p ≤ 0.005). UV, Fluor, and MFGE [RNA] are seemingly valid surrogates of cell/tissue ribosome content, although each method has advantages (e.g., ease of use) and disadvantages (e.g., magnitudes of bias) discussed herein. Finally, the RiboAb cocktail may also represent ribosome content, although this should be further explored in other models.