One step sybr primerscript rt pcr kit

The total RNA was extracted from cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and mature miR-185 expression was detected in cells using a Hairpin-it^™ miRNAs qPCR kit (Shanghai Genepharma Co., Ltd.). Thermocycling conditions included an initial step at 98°C for 1 min, and 40 cycles at 95°C for 5 sec and at 62–68°C for 15 sec, 72°C for 2 min and final extension at 72°C for 10 min. The expression of small nucleolar RNU6B was used as an endogenous control. SOX9, β-catenin and c-Myc expression was measured using a SYBR green qPCR assay (One Step SYBR PrimerScript RT-PCR Kit, Takara Biotechnology Co., Ltd., Dalian, China). Reverse transcription and amplification were performed at 42°C for 15 min, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec using an ABI Prism 7900 HT (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following primer sequences were used for the PCR: RNU6B, reverse 5′-AACGCTTCACGAATTTGCGT-3, forward 5′-CTCGCTTCGGCAGCACA-3; SOX9, forward 5′-AGCGAACGCACATCAAGAC-3′, reverse 5′-CTGTAGGCGATCTGTTGGGG-3′; β-catenin, forward 5′-AAAGCGGCTGTTAGTCACTGG-3′, reverse 5′-CGAGTCATTGCATACTGTCCAT-3′; c-Myc, forward 5′-GGCTCCTGGCAAAAGGTCA-3′, reverse 5′-CTGCGTAGTTGTGCTGATGT-3′; GAPDH, forward 5′-GGAGCGAGATCCCTCCAAAAT-3′, reverse 5′-GGCTGTTGTCATACTTCTCATGG-3′. Data were analysed using the 2^−ΔΔCq method (21 (link)).