Qiashredder mini column

Cells were lysed and homogenized with a QIAshredder mini column (QIAGEN, Germantown, TN, USA). Total RNA was extracted with Qiagen RNeasy mini kit. cDNA was prepared from 1 mg total RNA using the iScript Kit (Bio-rad, Hercules, CA, USA). RT-qPCR was performed with Thermo StepOne Plus Real-Time PCR System, and data were analyzed with the DataAssist^TM program. The primers were Runx2 forward: 5′-ACG AGG CAA GAG TTT CAC CTT GAC-3′, Runx2 reverse: 5′- AGG TAG CTA CTT GGG GAG GAT TTG-3′; ALP forward:5′- TGC GCA GGA TTG GAA CAT CAGT-3′, ALP reverse: 5′- TGC ACC CCA AGA CCT GCT TTAT-3′; osteopontin (OPN) forward 5′-TAC GAC CAT GAG ATT GGC AGT GA-3′, OPN reverse: 5′-TAT AGG ATC TGG GTG CAG GCT GTAA-3′. The primers for mouse β-actin as controls were forward 5‘-AGA GGG AAA TCG TGC GTG AC-3′ and reverse 5‘-CAA GAA GGA AGG CTG GAA AA-3′.

Cells were lysed and homogenized with a QIAshredder mini column (QIAGEN). Total RNA was extracted with Qiagen RNeasy mini kit. cDNA was prepared from 1 µg total RNA using the iScript Kit (Bio-rad). PCR amplification was performed in a 30 µl reaction with 1 µl cDNA reaction using Qiagen PCR kit according to the manufacturer's protocol. For comparison between the cells from WT and P2Y₂ KO mice, RT-PCR was performed and the products were analyzed by agarose gel electrophoresis. The primers for mouse osteocalcin were forward 5′-CAG GAG GGC AAT AAG GTA GT-3′ and reverse 5′-GAG GAC AGG GAG GAT CAA G-3′. The primers for mouse β-actin as controls were forward 5′-AGA GGG AAA TCG TGC GTG AC-3′ and reverse 5′-CAA GAA GGA AGG CTG GAA AA-3′.