Figure 2

Quantitative polymerase chain reaction (qPCR) validation of the transcriptome analysis of osteoclast cultures reveals seven genes differentially expressed in autosomal dominant osteopetrosis type II (ADO II) patients and healthy donors. (a) Tartrate-resistant acid phosphatase (TRAP) staining of 14-day cultures of osteoclasts from a healthy donor (left) and an osteopetrotic patient (right). No difference could be observed at the morphological level. The pictures shown here are representative of the observations made on the osteoclast cultures from 15 osteopetrotic patients and 31 healthy donors (scale bar: 50 μm). (b) The number of multinucleated and TRAP-positive osteoclasts was evaluated for all the control and osteopetrotic osteoclast cultures. No significant difference was observed between the two types of cultures. (c) The expression levels of three osteoclastic differentiation markers were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR) from all the osteopetrotic and healthy donor osteoclast cultures. No significant difference was observed between the two types of culture for the expression of TRAP, CTSK, or RANK. (d) CLCN7 expression (on the microarray) was not different in the osteopetrotic osteoclasts when compared with the healthy donor osteoclasts, even though the osteopetrotic osteoclasts carried a CLCN7 mutation. (e) Significant differences in the expression of ITGB5, CES1, PRF1, SERPINE2, WARS, GBP4 and UCHL1 in osteoclasts from healthy donors and osteopetrotic patients obtained by microarray. (f) Significant differences in the expression of ITGB5, CES1, PRF1, SERPINE2, WARS, GBP4 and UCHL1 in osteoclasts from healthy donors and osteopetrotic patients validated by qPCR. Healthy donors in white bars, n=31; osteopetrotic patients in black bars, n=15. *P<0.05, **P<0.005, and ***P<0.001.