## 4 Discussion
However, it is equally acknowledged that the field of ES research uses a vast array of parameters, different cell types, as well as varying modalities for delivering the stimulation to the cells such as DC ES, AC ES, a capacitive set-up, or even electromagnetic stimulation. Even a factor as seemingly small as the electrode material can have a large impact on the cellular response due to differences in faradic by-products [36]. Therefore, the results in this study only serve as an indication of how MSCs react in the system described. Any decisions to use PPIA and YWHAZ as a reference gene pair should be first validated in each specific ES set-up. Hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) is produced at the cathode during DC ES, and therefore, the levels of exposure per cell to the H\({}_{2}\)O\({}_{2}\) concentration would be elevated when fewer cells are present in the well. The reason for GAPDH's increase in expression at the lower seeding density of 5000 cells/cm\({}^{2}\) could be linked to findings that GAPDH is involved in regulating intracellular redox levels [37]. A possible explanation for the lack of stability of ACTB could be due to its role in cell migration [38]. MSCs in a direct current electric field migrate towards the anode at electric field strengths as low as 25 mV/mm [13]. Consequently, this could provide rationale for ACTB's involvement in the cellular response. All in all, 18S, while stable, has drawbacks due to the fact that it is expressed at much higher levels than the average gene of interest, appearing after 10-12 cycles. Due to the difference between the gene of interest and the reference gene being an exponent of 2 in fold change calculations, small differences are highly sensitive, and errors can be reduced by selecting a reference gene with a \(C_{T}\) value more similar to that of the genes of interest in the study. Moreover, due its single exon any primers targeting 18S cDNA could also amplify the corresponding genomic region. Thus, contaminating DNA might skew the findings unless removed prior to reverse transcription. Overall, comparing the findings from NormFinder, the \(\Delta\Delta C_{T}\) method, and BestKeeper has proven the value of using multiple platforms. As much as NormFinder identified ACTB as the least stable gene at both seeding densities, it consistently placed GAPDH as the second most stable gene. This exposes a flaw in solely relying upon NormFinder, as when the fold changes were calculated, GAPDH significantly increased in the 5000 cells/cm\({}^{2}\) group. The Pearson correlation coefficient analysis from BestKeeper ranked GAPDH mostly in the middle of the dataset. This further highlights the importance of selecting a stable reference gene pair. A poor reference gene has the potential of giving inaccurate gene expression results, which could confound and delay the advancement of knowledge within the research community. Thus, the value in carefully choosing the reference gene pairs used in gene expression studies cannot be underestimated. In summary, our study identified PPIA and YWHAZ as the most stable reference genes following ES. These two genes were similarly found to be the most stable following long-term MSC culture [19]. The \(\Delta\Delta C_{T}\) method showed an undeviating similarity in expression of both genes. This was further reinforced by the BestKeeper Pearson's correlation coefficient data, which ranked PPIA and YWHAZ as the best potential gene pair overall with a very highly positive and consistent relationship at both seeding densities. This finding was also replicated in primary MSCs, which provides confidence that the conclusions from this study are not only applicable to the immortalized MSCs, but to primary cells from a different donor. Due to the wide variation in parameters, conditions, electrical and temporal regimes applied by numerous research groups, this paper cannot state that PPIA and YWHAZ are unilaterally the most stable reference genes under ES.