7500 ht sequence detection system

Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using a PrimeScript™ RT Master Mix (TaKaRa, Japan) according to the manufacturer's protocols. Real-time quantitative PCR was performed using the standard protocols on an Applied Biosystems 7500 HT Sequence detection system using SYBR® Premix Ex Taq™ (TaKaRa, Japan). The following primers were used for qPCR analysis: UFL1, forward, 5′-TGTGGATCAGGTGGAAGCAT-3′ and reverse, 5′-TACAGCTGAAGCCTGTTTGC-3′; TNF-α, forward, 5′-CAAGTAACAAGCCGGTAGCC-3′ and reverse, 5′-CCCTGAAGAGGACCTGTGAG-3′; IL-6, forward, 5′-TGAGTGTGAAAGCAGCAAGG-3′ and reverse, 5′-AAGACCAGCAGTGGTTCTGA-3′; IL-1β, forward, 5′-AGTGCAAACTCCAGGACAGA-3′ and reverse, 5′-GATACCCAAGGCCACAGGAA-3′; and Bos taurus GAPDH, forward, 5′-CATGACCACTTTGGCATCGT-3′ and reverse, 5′-CCATCCACAGTCTTCTGGGT-3′. Gene expression data were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by employing an optimized comparative Ct (2^-ΔΔCt) value method.

Cells were treated with various concentrations (0, 0.3, 0.6, and 0.9 mM) of NEFA for 3, 6, 9, and 12 h for RT-qPCR. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The purity and quantity of total RNA were detected using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). DNase I and the Prime Script RT Master Mix kit (TaKaRa, Otsu, Japan) were used for DNA removal and cDNA synthesis, respectively. Real-time quantitative PCR was performed using standard protocols on an Applied Biosystem 7500 HT Sequence detection system using SYBR ^®Premix Ex Taq™ (TaKaRa, Otsu, Japan). The PCR system consisted of 10 μL SYBR Premix Ex Taq, 2 μL cDNA, 0.4 μL ROX Reference Dye II, 0.4 μL of each primer (10 µM), and 6.8 μL double distilled water in a total volume of 20 μL. The PCR program consisted of one cycle at 95 °C for 30 s, 40 cycles at 95 °C for 5 s, and 60 °C for 34 s, with fluorescence signal collection at 60 °C. The primer sequences were shown in Table 1. Gene expression data were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by employing an optimized comparative Ct (2^−ΔΔCt) value method.