Iscript rt qpcr sample prep reagent

Total RNA was extracted using the iScript™ RT-qPCR Sample Prep reagent (Bio-Rad), according to the manufacturer’s instructions. QuantiNova SYBR Green RT-PCR Kit (QIAGEN, Hilden, Germany) was used for qPCR, along with specific primers for 18S (FWD 5′-CGGCTACCACATCCACGGAA-3′, REV 5′-CCTGAATTGTTATTTTTCGTCACTACC-3′) PCSK9 (FWD 5′-CCTGCGCGTGCTCAACT-3′, REV 5′-GCTGGCTTTTCCGAATAAACTC-3′), ANGPTL3 (FWD 5′-GCCTGTTGGAGACTCAGATGG-3′, REV 5′-TAGCACCTTCTGTGCCTGGG-3′), FAS (FWD 5′-GCAAATTCGACCTTTCTCA-3′, REV 5′-GGACCCCGTGGAATGTCA-3′), ACLY (FWD 5′-TGCAAAGTGAAGTGGGGTGA-3′, REV 5′-TTTGGGGTTCAGCAAGGTCA-3′), ACC1 (FWD 5′-ATGTCTGGCTTGCACCTAGTA-3′, REV 5′-CCCCAAAGCGAGTAACAAATTCT-3′), PCSK9 (FWD 5′-CCTGCGCGTGCTCAACT-3′, REV 5′-GCTGGCTTTTCCGAATAAACTC-3′), SCD1 (FWD 5′-AAAGCGAGGTGGCCATGTTA-3′, REV 5′-TCATGCCTCAAAACTGCCCT-3′).
The analyses were performed with the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) with cycling conditions of 45 °C for 10 min, 95 °C for 5 min, and a repetition of 40 cycles at 95 °C for 5 s followed by 30 s at 60 °C. The data were expressed as Ct values and used for relative quantification of targets with ΔΔCt calculations. The ΔΔCt values were determined by multiplying the ratio value between the efficiency of specific primers and housekeeping 18S. The efficiency was calculated as ((10^(−1/slope)) − 1) × 100.