7500 fast rt qpcr system

Total RNA was extracted from MG63 and U2OS cells using TRIzol^® reagent (Roche Applied Science). cDNA was synthesized using the Takara reverse transcriptase kit (Takara Bio Inc.) according to the manufacturer's instructions. The reverse transcription steps were as follows: 3 cycles of 30°C for 10 min followed by 3 cycles of 50°C for 60 min followed by 95°C for 5 min and 4°C holding. The cDNA strand was amplified by RT-qPCR using SYBR-Green I (Toyobo Life Science) on a 7500 fast RT-qPCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 60 sec at 95°C followed by 40 cycles of 15 sec at 95°C, 15 sec at 60°C and 45 sec at 72°C. GAPDH and U6 were used as internal references for mRNA or miRNA, respectively. The primer sequences were as follows. CCAT2, forward 5′-CCCTGGTCAAATTGCTTAAC-3′ and reverse, 5′-TTATTCGTCCCTCTGTTTTATGG-3′; GAPDH, forward 5′-CCACATCGCTCAGACACC-3′ and reverse, 5′-ACCAGGCGCCCAATA-3′; miR-143, forward 5′-AGCGTGTGTCGTGGAGTC-3′ and reverse, 5′-TCGTGAGATGAAGCACTGTAG-3′; U6, forward 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; and FOS-Like antigen 2 (FOSL2), forward 5′-GAGAGGAACAAGCTGGCTGC-3′ and reverse, 5′-GCTTCTCCTTCTCCTTCTGC-3′. The results were analyzed by the 2^−ΔΔCq method (17 (link)).