Steponeplus real time pcr detection system

Total RNA was isolated from cultured cells using TRIzol reagent. A total of 2 micrograms of RNA was reverse-transcribed to cDNA using a PrimeScript RT Reagent kit (cat. no. RRO47A; Takara Bio, Inc.) according to the manufacturer's protocols. Gene expression was then analyzed in these samples using the Applied Biosystems Step One Plus™ Real-Time PCR Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR mixture contained the following: SYBR^® Premix Ex Taq™ II (2X) 2 µl, PCR forward primer (10 µM) 0.8 µl, PCR reverse primer (10 µM) 0.8 µl, ROX reference dye (50X) 0.4 µl, cDNA (taken from the RT product mixture) 2 µl, deionized H₂O 6 µl. The real-time PCR conditions were as follows: Activation at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 5 sec, primer annealing and extension at 60°C for 30 sec and a ramp up back to 95°C. The quantification cycle (Cq) values for each gene, normalized to the expression levels of GAPDH, were calculated by the 2^−∆∆Cq method (29 (link)). The respective forward and reverse primer sequences for real-time PCR were as follows: MDR1, 5′-TGCTCAGACAGGATGTGAGTTG-3′ and 5′-TAGCCCCTTTAACTTGAGCAGC-3′; GAPDH, 5′-CAGGGCTGCTTTTAACTCTG-3′ and 5′-GATTTTGGAGGGATCTCGC-3′; Gli2, 5′-CTCAAGGAAGATCTGGACAGG-3′ and 5′-GATGTGCTCGTTGTTGATGTG-3′; and Gli1, 5′-AGCGTGAGCCTGAATCTGTG-3′ and 5′-CAGCATGTACTGGGCTTTGAA-3′.