Oligo dt

The expression profiles of PDGF and IGF were measured using a one-step SYBR PrimeScript RT-PCR kit (Takara Bio, Inc., Otsu, Japan) in an ABI Prism 7500 (Applied Biosystems; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. An RNA reverse transcription kit, dNTP, RNase inhibitor and oligo-dT (all Tiangen Biotech Co., Ltd., Beijing, China) were used for RT-qPCR. The 20 µl reaction mixture contained 4 µl cDNA, 2 µl 10X buffer, 0.4 µl dNTP (10 mol/l), 0.4 µl primer mix and 0.5 U Taq polymerase and ddH₂O. The qPCR procedure was as follows: Initial denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec and elongation at 72°C for 45 sec, with a final elongation at 72°C for 10 min and short storage at 4°C. The primer sequences utilized in RT-qPCR were as follows: PDGF, forward, 5′-GATCCGCTCCTTTGATGATC-3′ and reverse, 5′-GTCTCACACTTGCATGCCAG-3′; IGF, forward 5′-AAGCCTACAAAGTCAGCTCC-3′ and reverse, 5′-CGTCTTGTTTCCTGCACTTC-3′; and β-actin, forward, 5′-TGGTGGGTATGGGTCAGAAGGACTC-3′ and reverse, 5′-CATGGCTGGGGTGTTGAAGGTCTCA-3′. All primers were synthesized by Invitrogen; Thermo Fisher Scientific, Inc. The relative expression was analyzed using the 2^−ΔΔCq method (6 (link)) and the expression of all transcripts were normalized to that of the housekeeping gene β-actin.