Table 1.
The reference genes chose in this study.
Table 2.
Primers used in this study.
Figure 1.
The values of geNorm M (A) and geNorm V (B) of 16 reference genes.
(A) The genes with lower geNorm M values were considered more stable and the gene with M value below 0.5 was accepted as appropriate reference genes. Thus, Ppia, Tbp, Hprt and Eef1a1 were acceptable reference genes in this study. (B) The optimal number of reference genes required for normalization was determined by pairwise variation (geNorm V value of n/n+1) and a value below 0.15 indicated the minimum number (n) of genes. In this study, a combination of two reference genes was sufficient for normalization since V2/3 was 0.113.
Figure 2.
Heatmap for individual reference genes expression.
The relative gene expression levels of all samples were depicted in color-code. The threshold cycle value of each sample was normalized against that of all the reference genes. Expression levels of all samples were normalized against those of 1 d GM group respectively and log2 transformed. Sixteen reference genes were sorted from up to down by their geNorm M value. Red color indicated that the expression level of gene was up-regulated compared to that of 1 d of GM group (the brighter the higher), whereas green color indicated vice versa (the brighter the lower).
Figure 3.
Relative expression of various genes normalized against different reference genes in ATDC5 cells.
(A) Col2; (B) Col1; (C) Sox9; (D) ColX. *P<0.05 represented the significant effects among different culture medium.
Figure 4.
Relative expression of sixteen candidate reference genes in ATDC5 cells.
Cells were cultured in different medium for 7 d. The data were calculated using ΔCt methods with normalization against Tbp. Error bars represented the standard error of 3 biological replicates. Note that the Y axis was on a log10 scale.
Figure 5.
Relative expression of cartilage-specific genes normalized against different reference genes in mMSCs.
Col2; (B) Sox9. *P<0.05 represented the significant effects among different culture time.