Kapa sybr fast qpcr master mix

After isolating total RNAs using REzol reagent (Protech Technology Enterprise CO., Ltd.) and reverse transcribing the mRNA to cDNA using a PrimeScript RT Reagent kit (Takara Bio, Inc.), the levels of HGF mRNA were detected using KAPA SYBR FAST qPCR Master Mix, according to the manufacturer's instructions. The MyGo PCR Detection System (IT-IS Life Science Ltd.) was used for detection. The following primers were used in the experiment: HGF forward, 5′-GAA-TGC-ATG-ACC-TGC-AAC-GG-3′ and reverse, 5′-TGT-CGG-GAT-ATC-TTT-CCG-GC-3′; and GAPDH forward, 5′-CAC-CCA-TGG-CAA-ATT-CCA-TGG-CA-3′ and reverse, 5′-TCT-AGA-CGG-CAG-GTC-AGG-TCC-ACC-3′. The RT-qPCR thermocycling conditions included an initial denaturation at 95°C for 180 sec, followed by 40 cycles with denaturation at 95°C for 10 sec, annealing at 60°C for 20 sec, and extension at 72°C for 30 sec. The relative level of gene expression was determined using the comparative C_q (2^−ΔΔCq) method (36 (link)), with GAPDH used as the normalization reference.

Total RNA was extracted from ccRCC cells using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) and complementary DNA was synthesized from RNA using the PrimeScript RT reagent kit (Takara Bio, Inc.). The following temperature protocol was used for reverse transcription: 37˚C for 50 min followed by 70˚C for 15 min. Subsequently, qPCR was performed using the Kapa SYBR^® FAST qPCR Master Mix (Takara Bio, Inc.) and was analyzed using the MyGo PCR detection system (IT-IS Life Science Ltd.). The thermocycling conditions were: 10 min initial denaturation at 94˚C, 15 sec denaturation at 94˚C and 30 sec of annealing at 55˚C (40 cycles) and final extension for 1 min at 72˚C. The mRNA expression levels of AKIP1, VEGFA, VEGFR2 and Rac1 were quantified using the 2^-∆∆Cq method (15 (link)) and were normalized to the internal reference gene GAPDH. The primer sequences used in the present study are as follows: AKIP1 forward, 5'-AGAACATCTCTAAGGACCTCTACAT-3' and reverse, 5'-TCCAGAATCAACTGCTACCACAT-3'; VEGFA forward, 5'-GGGCAGAATCATCACGAAGT-3' and reverse, 5'-TGGTGATGTTGGACTCCTCA-3'; VEGFR2 forward, 5'-CTCTTGGCCGTGGTGCCTTTG-3' and reverse, 5'-GTGTGTTGCTCCTTCTTTCAAC-3'; Rac1 forward, 5'-AAAATGTCCGTGCAAAGTGGT-3' and reverse, 5'-CTCGATCGTGTCTTTATCATCCC-3'; GAPDH forward, 5'-AATGGACAACTGGTCGTGGAC-3' and reverse, 5'-CCCTCCAGGGGATCTGTTTG-3'.