4.6. RNA Isolation and cDNA Synthesis

Cell-seeded collagen gels (six 0.5 mL-gels per experimental group) were removed from the transwell inserts and incubated with collagenase II solution (3 mg/mL collagenase II, 235 U/mg, Biochrom, Berlin, Germany in α-MEM, 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin, 3 mM CaCl₂) for 1 h at 37 °C. The digests were transferred to 15 mL tubes, washed with PBS and centrifuged. The pellets were washed with PBS and RNA was isolated from these pellets as well as from the osteoblast-seeded transwell membranes using a commercially available kit (peqGOLD MicroSpin Total RNA Kit, Peqlab, Erlangen, Germany). cDNA was generated using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, part of Thermo Fisher Scientific) according to manufacturer’s instructions.