Rt qpcr mrna sybr green detection kit

The expression of CCDC67 gene was detected by RT-qPCR. The primers of CCDC67 and GAPDH genes were synthesized by Shanghai GeneChem Co., Ltd., and the sequences were as follows: CCDC67 forward, 5′-GAGGATCCCCGGGTACCGGTCGCCACCATGGAGAACCAAGCCCATAATAC-3′ and reverse, 5′-TCACCATGGTGGCGACCGGTATGTGTCTATTTTGTTTTAGC-3′; GAPDH forward, 5′-TGAAGGTCGGAGTCAACGG-3′ and reverse, 5′-CTGGAAGATGGTGATGGGATT-3′. Total RNA was extracted from TPC-1 and TPC-1-Luc-Puromycin-CCDC67 cells (continuously cultured in puromycin-free medium for ≥4 weeks) using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and 1 µl total RNA was reverse-transcribed to cDNA using PrimeScript™ II kit (Takara Bio, Inc.) according to the manufacturer's protocol. The resulting cDNA was quantified using a RT-qPCR mRNA SYBR Green Detection kit (Takara Bio, Inc.). The resulting cDNA (2 µl) was used as the template for PCR in a 20-µl reaction volume containing 10 µl 2X SYBR Premix Ex Taq II, 0.8 µl each of 10 µmol/l forward and reverse primers and 6.4 µl ddH₂O. The thermocycling conditions were as follows: 5 sec at 95°C, followed by 50 cycles of 95°C for 5 sec, 60°C for 50 sec. The mRNA expression level of GAPDH was used for normalization. The threshold cycle (Cq) value was recorded, and the data were analyzed by the comparative 2^−ΔΔCq method (15 (link)).