2.8. Real-Time Quantitative PCR Analysis

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.