Reverse transcription reaction

The left femur bones were pulverized in liquid nitrogen with a mortar and pestle that had been pre-chilled in dry ice (23 (link)). Powdered bone was homogenized in One Step-RNA reagent (BioBasic, Canada) and RNA extraction was performed. The quality and quantity of the RNA samples were assayed spectrophotometrically, with the ratios of absorbance at 260 nm and 280 nm ranging from 1.8 to 2.0, and the integrity of the RNA preparations was examined by agarose gel electrophoresis. Complementary DNA (cDNA) was synthesized using 500 ng of RNA through a reverse transcription reaction (Takara Bio Inc., Tokyo, Japan) in proportion to the manufacturer's instructions. Real-time PCR was performed using a RotorGene 3000 instrument (Corbett Research, Australia). Primers used for OPG, RANK, RANKL, iNOS and β-actin are listed in Table 1. The mRNA levels of OPG, RANK, RANKL and iNOS were normalized to β- actin mRNA levels. PCR was run as 40 cycles at 95 °C for 15 s, 62 °C for 30 s, and 72 °C for 30 s. The negative controls for each target showed an absence of carryover. Data of target mRNA copies were calculated relative to β- actin using the 2^–ΔCt method.