Stably expressed reference genes during differentiation ofbone marrow-derived mesenchymal stromal cells

Human mesenchymal stromal cells (MSCs) from different parts of the body (i.e. bone marrow, BM) have distinct cellular and molecular features including global gene expression profiles. Quantitative polymerase chain reaction is a reliable method used in the quantification of gene expression. Correct assessment of target gene expression mostly depends on the reference gene (RG) of choice. Herein, expression levels of RGs (n = 19) in adipo-/osteogenic-differentiated and control (uninduced) BM-MSCs from donors were measured using the RealTime ready Human Reference Gene Panel. Characterization of BM-MSCs was assessed using in vitro differentiation by histochemical staining and immunophenotyping of cells by flow cytometric analysis. BM-MSCs successfully differentiated into adipogenic and osteogenic lineages. When three groups were analyzed together, the NormFinder and GeNorm software programs identified PBGD as the most stable RG. GeNorm also reported G6PDH as the other stable gene. Due to their low expression, PBGD and G6PDH were not suitable RG candidates. RPLP0 had slightly lower stability but very high expression, rendering it as the best RG. When either adipogenic- or osteogenic-induced MSCs were analyzed with control BM-MSCs, the most stable RGs with high expression levels were revealed to be GAPDH and RPLP0. Even though ACTB had the highest expression, its stability was low.