M mlv rt kit

Total RNA was extracted from tissues and cell lines using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed at 65°C for 120 sec using the M-MLV RT kit (Promega Corporation, Madison, WI, USA) following the manufacturer's protocol. The expression of miR-338-3p was quantified using TaqMan microRNA assays (Ambion; Thermo Fisher Scientific, Inc.) with the ABI 7900 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The mRNA expression of MACC1 was detected using SYBR^® Green qPCR SuperMix (Invitrogen; Thermo Fisher Scientific, Inc.) and quantified using ABI 7900 Sequence Detection System. Amplification was performed using the following thermocycling protocol: Preheating at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 5 sec and annealing/extension at 60°C for 20 sec. The primers used for the current study were as follows: miR-338-3p forward 5′-TGCGGTCCAGCATCAGTGAT-3′ and reverse 5′-CCAGTGCAGGGTCCGAGGT-3′; U6 forward 5′-GCTCGCTTCGGCAGCACA-3′ and 5′-GAGGTATTCGCACCAGAGGA-3′; MACC1 forward 5′-CCTTCGTGGTAATAATGCTTCC-3′ and reverse 5′-AGGGCTTCCATTGTATTGAGGT-3′; GAPDH forward 5′-ACCACAGTCCATGCCATCCAC-3′ and reverse 5′-TCCACCACCCTGTTGCTGTA-3′. The relative expression of miR-338-3p or MACC1 was normalized to U6 small nuclear RNA or GAPDH, respectively and calculated using the 2^−ΔΔCq method (22 ).