Retroscript reverse transcription kit

Total RNA from the tumor tissues was isolated using TRIzol (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol. RETROscript™ Reverse Transcription kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1 µg RNA were used for cDNA synthesis. SYBR Green One-Step qRT-PCR kit (Thermo Fisher Scientific, Inc.) and a 7900 HT Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) were used for qPCR. Data were normalized to the internal control GAPDH and presented as an expression fold change using the 2^−ΔΔCq method (16 (link)). Thermocycling conditions were as follows: 95°C for 3 min, followed by 40 cycles at 94°C for 30 sec, 60°C for 30 sec and 72°C for 60 sec. The gene-specific primers were selected according to the literature. The primers were synthesized by Sangon Biological Engineering Technology Services Co., Ltd. (Shanghai, China). The primer sequences were as follows: PI3K, forward 5′-CGTTTCTGCTTTGGGACAAC-3′ and reverse 5′-CCTGATGATGGTGGAG-3′; AKT, forward 5′-TGAGAGAAGCCACGCTGTC-3′ and reverse 5′-CGGAGAACAAACTGGATGAA-3′; VEGF, forward 5′-TAGACGTTCCCTGCCAGCAA-3′ and reverse 5′-AGCATCCGAGGAAAACATAAAATCTT-3′; and GAPDH, forward 5′-AACGGATTTGGTCGTATTGGG-3′ and reverse 5′-TCGCTCCTGGAAGATGGTGAT-3′.