Fig. 1
A Cq values are presented as quantification cycle (Cq, n = 3) as second derivate maximum of the fluorescence curve and are inversely proportional to the amount of target mRNA within 1 μg of total RNA retrieved from the cementoblasts. Cq expression values of candidate reference genes, overall are for specimens without treatment (n = 13*3 duplication) in cementoblasts. B Expression levels of candidate reference genes in periodontal ligament cells (n = 13*3 duplication) without treatment. Cq values exported with identical threshold setting (mean of three technical replicates). Boxplots represent the median (central horizontal line), the interquartile range (IQR, 25/75 quartile, box) and the data range (whiskers) without outliners and extreme values. Outliers and extreme values are defined as Cq values more than 1.5 and 3 times the IQR apart from the upper/lower quartile and are denoted as circles and asterisms respectively. C On OCCM-30 cells, the geNorm analysis of the expression stability values (M value) of the 13 candidate reference genes, for which specific primers could be constructed. Average expression stability values of overall (pooled) specimens derived by stepwise exclusion of the least stable reference gene across all specimens and experiment conditions (n = 13*3 duplication). A smaller M value indicates a more stable expression. The most stable genes are on the right and the least stable genes are on the left. D Pairwise variation (V) of the 13 candidate reference genes calculated by geNorm to determine the suitable number of reference genes for OCCM-30 cells for RT-qPCR data normalization in overall studies (n = 13*3 duplication). The threshold used was 0.15. E On periodontal ligament cells, the geNorm analysis of the expression stability of the 13 candidate reference genes tested. Overall average expression stability values (M) derived by stepwise exclusion of the least stable reference gene across all specimens and experiment conditions. A higher M indicates a less gene expression. F Determination of the suitable number of reference genes for RT-qPCR data normalization on periodontal ligament cells. The geNorm calculation by the pairwise variation (V) indicates that V values lower than 0.15 indicated a sufficient normalization can be achieved