Mx3000 platform

Total RNA was extracted with TRIzol (Invitrogen, Carlsbad, USA) and then converted to cDNA with Hifair II 1st Strand cDNA Synthesis Kit (Yeasen, Shanghai, China) following the kit instructions. SYBR green PCR Master Mix (Takara, Dalian, China) was used for real-time qPCR on an MX 3000 platform (Agilent, Boeblingen, Germany). The primers for real-time qPCR were as follows: Runx2, 5’-ATAGCAAAGGCCCTCACTAA-3’ (forward) and 5’-AACTGGCTCTTCTGCTGATT-3’ (reverse); Col 1, 5’-GAGGCATAAAGGGTCATCGTGG-3’ (forward) and 5’-CATTAGGCGCAGGAAGGTCAGC-3’ (reverse); OC, 5’-TGACCTCACAGATGCCAAGC-3’ (forward) and 5’- CGCCGGAGTCTGTTCACTAC-3’ (reverse); and GAPDH, 5’- ACCACAGTCCATGCCATCAC-3’ (forward) and 5’-TCCACCACCCTGTTGCTGTA-3’ (reverse).
The expression levels of each target gene were normalized to the corresponding GAPDH threshold cycle (CT) values using the 2−▵▵CT comparative method [16 (link)].

RNA was extracted using Trizol (Sigma) and equal amounts of RNA were added to a reverse-transcriptase reaction mix (m-MLV, Promega). Real time PCR was performed on an Mx3000 platform (Agilent) using the FastStart Universal Probe Master kit (ROX, Roche) and run for 40 cycles. Specificity of the reactions was determined by subsequent melting curve analysis. Stratagene analysis software was used to remove background fluorescence [66 (link)]. The number of cycles needed to reach the crossing point for each sample was used to calculate the amount of each product using the 2^{−Δ Δ Ct} method. The levels of PCR product were expressed as a function of actin. The primers used are listed in Supplementary Table 1.

Total RNA was isolated by TRIzol® (Invitrogen). Equal amounts of RNA were reverse transcribed using a reverse-transcriptase reaction mix (M-MuLV, Promega, Madison, WI). Real-time PCR was performed on the Mx3000 platform (Agilent Technologies Inc., Santa Clara, CA) using the KAPA SYBR FAST qPCR Master Mix (Sigma-Aldrich, St. Louis, MO). mRNA levels were calculated by the 2^–ΔΔCt method. Primer sequences are included in Table S1 as Additional File 1.