Primary osteoblast cells were isolated by 10-minute sequential digestion of 2 day-old (specific pathogen free) neonatal C57BL/6 calvaria with 0.25 U/ml collagenase D (Roche Diagnostics, Indianapolis, IN, USA) and 2.2 U/ml dispase (Invitrogen, Carlsbad, CA, USA). Digest fractions 2 to 6 were pooled as primary calvarial osteoblasts. Osteoblasts were cultured in complete MEM media supplemented with 10% heat-inactivated FBS, 20 U/mL penicillin, 20 ug/mL streptomycin and 2 mM L-glutamine (Invitrogen). Osteoblasts were differentiated in complete BGJb media supplemented with 10% heat-inactivated FBS, 20 U/mL penicillin, 20 ?g/mL streptomycin and 2 mM L-glutamine (Invitrogen) with 50 ?g/ml of ascorbate acid (Sigma-Aldrich, St Louis, MO, USA) and 10 mM ?-glycerophosphate (Sigma-Aldrich) from day 7 of culture. Cells were harvested on day 5 (proliferation phase), day 14 (differentiation phase) and day 21 (mineralization phase) representing different stages of osteoblast differentiation and RNA was extracted using an RNeasy kit (Qiagen), according to manufacturer’s instructions.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from cell pellets using the RNeasy kit (Qiagen, Valencia, CA, USA).
Label
biotin
Label protocol
5 ug total RNA was used in a standard Affymetrix single amplification protocol.
Hybridization protocol
standard Affymetrix procedures except for use of custom GNF chip washer
Scan protocol
standard Affymetrix procedures using GCS3000 scanner
