RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation

Figure 1 Isolated dental pulp stem cells and validation of osteogenic differentiation by alizarin red staining.

Figure 2 Gene expression stability by comprehensive ranking (RefFinder). A: Overall analysis (all samples); B: Subset 1 (clonogenic medium under 35 d); C: Subset 2 (osteogenic medium under 35 d); D: Subset 3 (clonogenic and osteogenic media from 1 to 21 d); E: Subset 4 (clonogenic and osteogenic media from 21 to 35 d); F: Subset 5 (only osteogenic medium from 1 to 21 d); G: Subset 6 (only osteogenic medium from 21 to 35 d); H: Subset 7 (only clonogenic medium from 1 to 21 d); I: Subset 8 (only clonogenic medium from 21 to 35 d). ACTB: Actin beta; ALAS1: Aminolevulinic acid synthase 1; B2M: Beta-2-microglobulin; EF1α: Eukaryotic translational elongation factor 1 alpha; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; RPLP0: Ribosomal protein, large, P0; SDHA: Succinate dehydrogenase complex, subunit A, flavoprotein; TBP: TATA-binding protein; TUB: Tubulin; YWHAZ: Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta.

Figure 3 Pairwise variation calculated by geNorm.

Figure 4 Effect of the reference gene (glyceraldehyde-3-phosphate dehydrogenase or TATA-binding protein/ribosomal protein, large, P0) on the gene expression of RUNX2. A: Normalization data of all samples (from the three donors) under clonogenic or osteogenic media by glyceraldehyde-3-phosphate dehydrogenase (GAPDH); B: Normalization data of all samples (from the three donors) under clonogenic or osteogenic media by TATA-binding protein (TBP)/ribosomal protein, large, P0 (RPLP0); C: Normalization data of only donor#1 under clonogenic or osteogenic media by GAPDH; D: Normalization data of only donor #1 under clonogenic or osteogenic media by TBP/RPLP0; E: Normalization data of only donor #2 under clonogenic or osteogenic media by GAPDH; F: Normalization data of only donor #2 under clonogenic or osteogenic media by TBP/RPLP0; G: Normalization data of only donor #2 under clonogenic or osteogenic media by GAPDH; H: Normalization data of only donor #3 under clonogenic or osteogenic media by TBP/RPLP0. RPLP0: Ribosomal protein, large, P0; TBP: TATA box binding protein; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; RUNX2: Runt-related transcription factor 2.