4.4. Real-Time Reverse Transcriptase (RT)-Polymerase Chain Reaction (PCR)

RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. For mRNA anaylsis, 500 ng of RNA were reverse transcribed into cDNA using the Quantitect Reverse Transcription Kit (Qiagen, Hilden, Germany) and a cycler (TC-3000, Techne, Bibby Scientific, Burlington, NJ, USA). The mRNA expression of cathepsin K (CtsK) and calcitonin receptor (CALCR) was analyzed by real-time RT-PCR using the Roche LightCycler^® FastStart DNA Master^Plus SYBR Green I (Roche, Basel, Switzerland) and a LightCycler^® 2.0 instrument (Roche). Primers used are listed in Table 1. The cycling procedure started with heating for 10 min at 95 °C, followed by 40 cycles of denaturation for 5 s at 95 °C, annealing for 5 s at 60 °C and elongation for 5 s at 72 °C. Purity of the RT-PCR products was verified by a melting curve. Samples that were not reverse transcribed or received water instead of cDNA served as negative controls. Results were evaluated using delta threshold cycle (CT) values and referenced to the housekeeping gene beta-2-microglobulin (B2M).

Human primers used for real-time RT-PCR analysis.

for: forward primer; rev: reverse primer.