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 electrop...
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Wiley
2025-01-01
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| Series: | Physiological Reports |
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| Online Access: | https://doi.org/10.14814/phy2.70173 |
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| author | Joshua S. Godwin J. Max Michel C. Brooks Mobley Gustavo A. Nader Michael D. Roberts |
| author_facet | Joshua S. Godwin J. Max Michel C. Brooks Mobley Gustavo A. Nader Michael D. Roberts |
| author_sort | Joshua S. Godwin |
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| description | 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. |
| format | Article |
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| institution | Kabale University |
| issn | 2051-817X |
| language | English |
| publishDate | 2025-01-01 |
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| spelling | doaj-art-12fb2df2773545e3ad31f2104657f8a32025-01-15T13:36:31ZengWileyPhysiological Reports2051-817X2025-01-01131n/an/a10.14814/phy2.70173Skeletal muscle ribosome analysis: A comparison of common assay methods and utilization of a novel RiboAb antibody cocktailJoshua S. Godwin0J. Max Michel1C. Brooks Mobley2Gustavo A. Nader3Michael D. Roberts4School of Kinesiology Auburn University Auburn Alabama USASchool of Kinesiology Auburn University Auburn Alabama USASchool of Kinesiology Auburn University Auburn Alabama USADepartment of Kinesiology and Huck Institutes of the Life Sciences The Pennsylvania State University University Park Pennsylvania USASchool of Kinesiology Auburn University Auburn Alabama USAAbstract 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.https://doi.org/10.14814/phy2.70173electrophoresisfluorometryRiboAbribosome pelletingtotal RNAUV–Vis |
| spellingShingle | Joshua S. Godwin J. Max Michel C. Brooks Mobley Gustavo A. Nader Michael D. Roberts Skeletal muscle ribosome analysis: A comparison of common assay methods and utilization of a novel RiboAb antibody cocktail Physiological Reports electrophoresis fluorometry RiboAb ribosome pelleting total RNA UV–Vis |
| title | Skeletal muscle ribosome analysis: A comparison of common assay methods and utilization of a novel RiboAb antibody cocktail |
| title_full | Skeletal muscle ribosome analysis: A comparison of common assay methods and utilization of a novel RiboAb antibody cocktail |
| title_fullStr | Skeletal muscle ribosome analysis: A comparison of common assay methods and utilization of a novel RiboAb antibody cocktail |
| title_full_unstemmed | Skeletal muscle ribosome analysis: A comparison of common assay methods and utilization of a novel RiboAb antibody cocktail |
| title_short | Skeletal muscle ribosome analysis: A comparison of common assay methods and utilization of a novel RiboAb antibody cocktail |
| title_sort | skeletal muscle ribosome analysis a comparison of common assay methods and utilization of a novel riboab antibody cocktail |
| topic | electrophoresis fluorometry RiboAb ribosome pelleting total RNA UV–Vis |
| url | https://doi.org/10.14814/phy2.70173 |
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