Selection and reliability of internal reference genes for quantitative PCR verification of transcriptomics during the differentiation process of porcine adult mesenchymal stem cells

Introduction: The objective of this study was to find highly reliable internal-control genes (ICGs) for normalization of qPCR data from porcine adult mesenchymal stem cells induced to differentiate toward adipogenic and osteogenic lineages. Methods: Stem cells were acquired from subcutaneous back fat and bone marrow of three castrated Yorkshire crossbred male pigs. Adipose and bone marrow-derived stem cells (ADSCs and BMSCs) were cultured in vitro with specific osteogenic or adipogenic differentiation medium for 4 weeks. Total RNA was extract for microarray (13,000 oligonucleotides) and qPCR analyses. Microarray data were used to uncover the most stably expressed genes (that is, potential ICGs). Co-regulation among potential ICGs was evaluated with Ingenuity Pathway Analysis. qPCR was performed on the non-coregulated ICGs candidates and on specific osteogenic (COL1A1) and adipogenic (DBI) genes. geNorm was used to uncover the most reliable ICGs by using qPCR data and the optimal number of ICGs to be used to calculate the normalization factor. Results: Microarray data analysis revealed 27 potential ICGs. Among those, 10 genes without known co-regulation were selected to perform qPCR. geNorm performed on qPCR data uncovered high stability in expression ratio among the selected ICGs. However, especially reliable normalization was obtained by geometric mean of NSUN5, TIMM17B, and VPS4A. The effect of normalization, assessed on specific osteogenic (COL1A1) and adipogenic (DBI) genes, was apparent for the adipogenic and less apparent for the osteogenic differentiation. Conclusions: The combination of microarray data and pairwise gene analysis allowed identification of novel and highly reliable ICGs for qPCR data normalization of adult porcine stem cells induced to differentiate to adipogenic and osteogenic lineages.