Faststart universal sybr premix extaq 2

We isolated total RNA from BMSCs by TRIzol reagent (Life Technologies, USA) and synthesized complementary DNAs (cDNAs) from total RNA. Then, we performed quantitative reverse transcriptase PCR (qRT-PCR) by FastStart Universal SYBR Premix ExTaqTM II (Takara Biotechnology, Japan) on an ABI Prism 7900 HT Sequence detection system (Applied Biosystems, USA). We used 100 μl RNA in RT-PCR experiments. The reaction conditions were as follows: 95°C, 30 s; 95°C, 10 s; 60–65°C, 10 s; for 40 cycles. The primer sequences used for qRT-PCR were as follows: Alp: forward, 5′-CCA ACT CTT TTG TGC CAG AGA-3′ and reverse, 5′-GGC TAC ATT GGT GTT GAG CTT TT-3′; Ocn: forward, 5′- CTG CGC TCT GTC TCT CTG AC-3′ and reverse, 5′- TTA AGC TCA CAC TGC TCC CG-3′; OPG: forward, 5′- AGT GTG AGG AAG GGC GTT AC-3′ and reverse, 5′-AAT GTG CTG CAG TTC GTG TG-3′; Runx2: forward, 5′-AAA TGC CTC CGC TGT TAT GAA-3′ and reverse, 5′-GCT CCG GCC CAC AAA TCT-3′.

Total RNA was isolated using TRIzol Reagent (Takara) according to the manufacturer's instructions. The concentration of RNA was determined by measuring the optical density at wavelengths of 260 nm and 280 nm. 1 μg of the total RNA was used to generate cDNA with the First Strand cDNA Synthesis kit (Bimake). Then, qPCR was performed using FastStart Universal SYBR Premix ExTaqTM II (Takara Biotechnology) on an ABI PRISM^® 7900HT System (Applied Biosystems). The relative gene expression was analysed by the relative standard curve method (2^−ΔΔCT) with Gapdh as the reference. The primers used for qPCR are listed in Table S1.

Total RNA of cultured cells was extracted using TRIzol reagent (Thermo Scientific) following the manufacturer’s instructions. One microgram of total RNA was then reverse-transcribed into complementary DNA using All-in-One cDNA Synthesis SuperMix (Biotool, USA). qRT‒PCR was performed with FastStart Universal SYBR Premix ExTaqTM II (TaKaRa Biotechnology, Japan) on an FTC-3000 real-time PCR system (Funglyn Biotech, Canada). Relative expression was calculated for each gene by the 2^−ΔΔCT method, and GAPDH amplification was used as an internal normalization. Sequences of the primers used for each gene are listed in Supplementary Table 1.

Total RNA from bone tissues or cultured cells was isolated using TRIzol reagent (Life Technologies, USA) and complementary DNAs (cDNAs) were synthesized from 1 μg of total RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, USA). Then, quantitative reverse transcriptase PCR (qRT-PCR) was performed using FastStart Universal SYBR Premix ExTaq^TM II (Takara Biotechnology, Japan) on an ABI Prism 7900 HT Sequence detection system (Applied Biosystems, USA). The primers for mRNAs were purchased from Sangon Biotech Co., Ltd, Shanghai. The primer sequences used for qRT-PCR were as follows: Alp: forward, 5′-CCA ACT CTT TTG TGC CAG AGA-3′ and reverse, 5′-GGC TAC ATT GGT GTT GAG CTT TT-3′; BGLAP: forward, 5′-CTG ACC TCA CAG ATG CCA AGC-3′ and reverse, 5′-TGG TCT GAT AGC TCG TCA CAA G-3′; HDAC7: forward, 5′-TGA AGA ATG GCT TTG CTG TG-3′ and reverse, 5′-CAC TGG GGT CCT GGT AGA AA-3′; mmu-miR-143–3p: forward, 5′-GGG GTG AGA TGA AGC ACT G-3′ and reverse, 5′-CAG TGC GTG TCG TGG AGT-3′; GADPH: forward, 5′-CAC CAT GGA GAA GGC CGG GG-3′ and reverse, 5′-GAC GGA CAC ATT GGG GGT AG-3′.

The subchondral bones dissected from the proximal tibias, chilled in liquid nitrogen, were cut into pieces. We used TRIzol reagent (Invitrogen, Carlsbad, CA) for the homogenization of the specimens, and then, the total RNA was harvested. One milligram of total RNA was employed to synthesize cDNA using a cDNA Synthesis kit (Fermentas, Burlington, Canada). After that, FastStart Universal SYBR Premix ExTaqTM II (TaKaRa Biotechnology, Japan) was employed to conduct qRT‒PCR. The 2^−△△CT approach was employed to measure relative gene expression.