Onestep rt pct kit

Total RNA of GCs was extracted using the RNeasy/miRNeasy Mini kit (Qiagen Benelux BV), according to the manufacturer's instructions. Total RNA (2 ng) was used for reverse transcription using the OneStep RT-PCT kit (Qiagen Benelux BV), following the manufacturer's instructions. The primers for miR-132 were the exact sequence of mature miR-132. U6 was used as the internal control. The primers were purchased from Qiagen Benelux BV. The thermocycling conditions for miRNA were as follows: 95°C for 15 min, followed by 95°C for 15 sec, and 60°C for 1 min (40 cycles). For the mRNA expression of Foxa1, GAPDH was used as the internal control. The primer sequences for Foxa1 and GAPDH were as follows: Foxa1 sense, 5′-AGGGCTGGATGGTTGTATTG-3′ and antisense, 5′-GCCTGAGTTCATGTTGCTGA-3′; GAPDH sense, 5′-GAAGGTGAAGGTCGGAGTC-3′ and antisense, 5′-GAAGATGGTGATGGGATTTC-3′. The thermocycling conditions for Foxa1 were as follows: 95°C for 5 min; 35 cycles of 95°C for 1 min, and 60°C for 1 min and 72°C for 1 min; and then 72°C for 7 min. RT-qPCR was performed in triplicate using a SYBR^® Premix Ex Taq Kit (Takara Bio, Inc.), according to the manufacturer's protocol, on a CFX96 Real-time PCR system (Bio-Rad Laboratories, Inc.). All reactions were run in triplicate and gene expression was determined using the 2^−∆∆Cq method (17 (link)).