Assessment of Reference Genes for Real-Time Quantitative PCR Gene Expression Normalization During C2C12 and H9c2 Skeletal Muscle Differentiation

Abstract

Skeletal muscle differentiation occurs during muscle development and regeneration. To initiate and maintain the differentiated state, a multitude of gene expression changes occur. Accurate assessment of these differentiation-related gene expression changes requires good quality template, but more specifically, appropriate internal controls for normalization. Two cell line-based models used for in vitro analyses of muscle differentiation incorporate mouse C2C12 and rat H9c2 cells. In this study, we set out to identify the most appropriate controls for mRNA expression normalization during C2C12 and H9c2 differentiation. We assessed the expression profiles of Actb, Gapdh, Hprt, Rps12 and Tbp during C2C12 differentiation and of Gapdh and Rps12 during H9c2 differentiation. Using NormFinder, we validated the stability of the genes individually and of the geometric mean generated from different gene combinations. We verified our results using Myogenin. Our study demonstrates that using the geometric mean of a combination of specific reference genes for normalization provides a platform for more precise test gene expression assessment during myoblast differentiation than using the absolute expression value of an individual gene and reinforces the necessity of reference gene validation.

Three‐dimensional spherical gelatin bubble‐based scaffold improves the myotube formation of H9c2 myoblasts

“…For the induction of differentiation, H9c2 cells on 2D gelatincoating substrates were first cultured in GM for 24 hr. The medium was then changed to low-serum differentiation medium (DM; DMEM supplemented with 1% FBS) and cultured for 14 days for the differentiation experiments (Masilamani, Loiselle, & Sutherland, 2014). The DM was changed every 2-3 days.…”

Involvement of oxidative stress and cytoskeletal disruption in microcystin-induced apoptosis in CIK cells

“…Both GAPDH and 18S rRNA were stable and unaffected by MC-LR exposure, similar to the results reported in HepG2 cells (Žegura et al, 2006, 2008) and carp (Jiang et al, 2013), therefore both of them were used as the endogenous assay control. The geometric mean of the expression level of GAPDH and 18S rRNA was used to normalize the RT-qPCR data (Vandesompele et al, 2002;Goossens et al, 2005;Nielsen and Boye, 2005;Masilamani et al, 2014).…”

Identification and Validation of Quantitative PCR Reference Genes Suitable for Normalizing Expression in Normal and Dystrophic Cell Culture Models of Myogenesis

“…Selection of an appropriate set of reference genes under such a varied suite of conditions is thus non-trivial, yet nevertheless critical for accurate measurement of gene expression during myogenesis. A number of studies have identified candidate reference genes for use in studies of mature muscle (and in cultured, terminally-differentiated myotubes) in a range of species8 , 9 , 10 , 11 , 12 , though candidates for normalization of gene expression throughout the myogenic program (particularly in cell culture) are more limited13 , 14, and are generally specific to a single cell line. Furthermore, such studies typically use at most 5 or 6 candidate genes (occasionally as few as 3), and do not necessarily validate the candidates identified against multiple genes with known expression patterns.…”