2.11. Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR)

Bone tissues or cells in the logarithmic growth phase were collected. The total RNA was extracted using the TRIzol method (Invitrogen) and reserved at −80°C. Total RNA was reversely transcribed into complementary DNA (cDNA) using PrimeScript@RT reagent kit (Perfect Real Time; TaKaRa) and reserved at −20°C for subsequent experiments. RT‐qPCR was performed using ABI7500 quantitative PCR system (ABI) with β‐actin as an internal reference. Reaction conditions were as follows: pre‐denaturation at 95°C for 5 minutes, 40 cycles of denaturation at 90°C for 30 seconds, annealing at 60°C for 40 seconds and extension at 72°C for 40 seconds. The primer sequences are shown in Table ​Table1.1. Each sample was repeatedly measured 3 times. PCR results were analysed using Opticon Monitor 3 software (Bio‐Rad). Each sample was repeatedly measured 3 times. The relative expression of the ratio of related genes to internal reference β‐actin was calculated using the 2^−ΔΔCt method, and then, the statistical results were obtained.

Primers used for RT‐qPCR analysis

Abbreviations: CST5, cystatin D; F, forward; OPG, osteoprotegerin; R, reverse; RANK, receptor activator of nuclear factor‐κB; RANKL, receptor activator of nuclear factor‐κB ligand; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction.