Quantitative real-time PCR (qRT-PCR)

Total RNA of MCs was extracted by TRIzol reagent (TaKaRa) according to the manufacturer's protocol. The cDNA was synthesized by a PrimeScript RT reagent Kit (TaKaRa) according to the manufacturer's protocol. All the primers, including cyclin D1 (forward, 5′-CGC CCT CCG TTT CTT ACT TC-3′ and reverse, 5′-GCA GTC AGG GGA ATG GTC T-3′), cyclin A2 (forward, 5′-AAG ATG CCC TGG CTT TTA GTG-3′ and reverse, 5′-TAACATTCACTGGCTTTTCGTCT-3′), Cyclooxygenase-2 (forward, 5′-AGGACTCTGCTCACG AAGGA-3′ and reverse, 5′-TGACATGGATTGGAACA GCA-3′), Microsomal PGE synthase-1 (forward, 5′-AGCACACTGCTGGTCATCAA-3′ and reverse, 5′-CTCCACATCTGGGTCACTCC-3′) and GAPDH (forward, 5′-GTCTTCACTACCATGGAGAAGG-3′ and reverse, 5′-TCATGGATGACCTTGGCCAG-3′) were designed using Primer 5 software (available at http://frodo.wi.mit.edu/) and synthesized by Invitrogen. qRT-PCR was performed with SYBR Premix Ex Taq (TaKaRa) and detected by ABI 7500 Real-Time PCR Detection System (Foster City, CA). The cycling program consisted of a preliminary denaturation (95°C for 10 min), followed by 40 cycles (95°C for 15 s and 60°C for 1 min). Fold changes in expression of each gene mRNA was normalized to GAPDH and analyzed using the delta-delta Ct method.