The broadly accepted method used to normalize gene expression through RT-PCR technology involves the expression of endogenous HKG. However, the utility of HKG must be validated for specific experimental conditions, since the expression of these endogenous genes can vary depending on experimental conditions 8–10, 15. In vitro systems, including cultured MCs, constitute an useful model to study many pathophysiological states affecting the glomeruli, such as glomerulosclerosis 1. Therefore, we aimed to determine the most stable reference genes for mRNA quantification in studies performed in vitro, mimicking the in vivo glomerular fibrosis using MMCs treated with TGF-β 10,13,15,30.
Since each algorithm ranked the best candidate HKG, the software packages recommended Ppia, Gapdh and 18S as the most stable reference genes between the groups. Peptidylprolyl isomerase A (Ppia), a highly abundant protein in the cytoplasm, takes part in various intracellular functions, including a homeostatic role in protein folding, protein trafficking, intracellular signaling, transcription, inflammation, apoptosis, and regulation of activity of other proteins 31–33. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) catalyzes the sixth reaction of anaerobic glycolysis, which produces ATP and pyruvate. Other than metabolic functions, this enzyme has been implicated in non-metabolic processes, such as apoptosis induction, DNA repair, cellular proliferation, and transcriptional activation 34–36. Small subunit 18S ribosomal RNA (18S) is the small component of eukaryotic cytoplasmic ribosomes and is one of the molecular markers 37,38.
The other two genes considered in this study (Hprt and Actb) are also known as common reference genes. Hypoxanthine phosphoribosyltransferase (Hprt) is responsible for purine metabolism, and deficiency of this gene dysregulates cell cycle-controlling functions and cell proliferation mechanisms 39,40. Actin beta (Actb), which is highly abundant in eukaryotic cells, is essential for a variety of cellular functions and is involved in maintaining the cell’s structure, integrity, and motility 41. Although extensively used as reference genes 14, Hprt and Actb ranked as the least stable in this study; however, further studies are needed to better delineate the interactions of these genes with TGF-β.
Since Ppia, Gapdh, and 18S were the most suitable candidate reference genes, we normalized them by each other, resulting in no statistically significant differences between groups, which suggests that these genes are good choices for our experimental conditions. After determining the candidate HKG by their stability values, we established the optimal number of reference genes using GenEx software. According to calculated Acc.SD, the optimal number of HKG in this model is the combination of two genes. When used together, Ppia and Gapdh showed a strong correlation, indicating that all samples were correlated and validating the best pair of HKG.
It is well demonstrated that TGF-β stimulates production of fibronectin, vimentin, and α-SMA in cultured MCs 2,42; thus, the best HKG combinations obtained in the present analysis were used to normalize these target genes. Several studies have reported that Ppia 14,17,43−48, Gapdh 44,49,50, and 18S 45,51−53 are suitable reference genes and could be used as normalizers of target genes in different models. In the present study, the top three candidate reference genes, whether used alone or in combination, showed the expected increase in the expression of the target genes in the TGF-β-treated group. In contrast, the less stable HKG, employed alone or in combination, did not yield these expected differences, indicating that the in silico analysis selected the better, more stable HKG for this in vitro fibrosis model; they also revealed that an inadequate choice of the endogenous standard HKG could influence the results.
It is important to mention that other non-tested genes in the preset study can also be used for normalization of the expression of target genes, and additional studies are needed to identify additional candidate genes. Furthermore, this study is specific to MMCs stimulated with TGF-β and, thus, the conclusions drawn from our study are not transferable to other models that employ MMCs.
Among the reference genes tested in this study, the combination of Ppia and Gapdh was the best HKG pair and should, therefore, be used as HKG in gene expression analysis in TGF-β-treated MMCs models.