One step tb green primescript rt pcr kit

Total RNA was extracted from cultured cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. miR-190b expression levels were detected using an ABI 7500 (Applied Biosystems Thermo Fisher Scientific, Inc.) using One Step TB Green^® PrimeScript™ RT-PCR Kit (Takara Bio, Inc.) after transcribing the extracted RNA into complementary DNA (cDNA) using MMLV Reverse Transcriptase First Strand cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 15 min, 85°C for 5s, and 4°C for 60 min. The sequences of the primers used were as follows: miR-190b forward, 5′-GGGTGATATGTTTGATAT-3′ and reverse, 5′-CAGTGCGTGTCGTGGAGT-3′; U6 small nuclear RNA forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; FOXP2 forward, 5′-AACAACAGCAGGCTCTCCAG-3′ and reverse, 5′-GGCACCTGCAGTGGTCTC-3′; and GAPDH forward, 5′-GGTGGTCTCCTCTGACTTCAACA-3′ and reverse, 5′-GTTGCTGTAGCCAAATTCGTTGT-3′. Expression levels of miR-190b or FOXP2 were calculated using the 2^−∆∆Cq method with U6 snRNA or GAPDH as endogenous controls (11 (link)). Experiments were performed in triplicate. The thermocycling conditions were as follows: Initial denaturation at 95°C for 30s, followed by 40 cycles of 95°C for 10s and 56°C for 30s.