Takara primescript kit

Total RNA from tissues and cell lines was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. RT was conducted using the Takara PrimeScript kit (Takara Biotechnology Co., Ltd.) at 37°C for 15 min. Subsequently, the RT-qPCR assay was performed using the ViiATM 7 Real-Time PCR system (Thermo Fisher Scientific, Inc.) with SYBR-Green Master Mix to detect gene expression levels. The following amplification conditions were used: Pre-denaturation at 95°C for 15 sec, followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 20 sec and extension at 72°C for 40 sec. Relative gene expression was calculated using the 2^−ΔΔCq method (24 (link)). GAPDH and U6 were set as an internal control. The primer sequences were as follows: HOXA-AS2 forward, 5′-CCCGTAGGAAGAACCGATGA-3′ and reverse, 5′-TTTAGGCCTTCGCAGACAGC-3′; miR-519 forward, 5′-CATGCTGTGACCCTCCAAAG-3′ and reverse, 5′-GAGAAAACAAACAGAAAGCGCT-3′; PD-L1 forward, 5′-TGCGGACTACAAGCGAATCA-3′ and reverse, 5′-GATCCACGGAAATTCTCTGGTT-3′; HIF-1α forward, 5′-ACTTGGACGCTCTGCCTATG-3′ and reverse, 5′-TTGCGGGGGTTGTAGA-3′; GAPDH forward, 5′-GAAGAGAGAGACCCTCACGCTG-3′ and reverse, 5′-ACTGTGAGGAGGGGAGATTCAGT-3′; and U6 forward, 5′-CTCGCTTCGGCAGCACATATACTA-3′ and reverse, 5′-ACGAATTTGCGTGTCATCCTTGCG-3′.