RNA Extraction and Real-Time PCR Analysis

After the fatty acid treatments, cells were directly lysed in the 25-cm² flasks using RLT lysis buffer (Qiagen, Chadstone, Victoria, Australia), cell lysates were collected, and total RNA extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer instructions. cDNA was synthesised starting from 0.5 μg of total RNA as a template and using SuperScript II (Life Technologies Australia Pty. Ltd.) as described previously (40 (link)). Real-time PCR to assess the expression of IL-6 and ribosomal protein S18 was carried out using CFX Connect 96 real-time PCR detection system (Bio-Rad) employing a two-step cycling program of 95°C for 20 s, and then 40 cycles at 95°C for 3 s and 60°C for 30 s. Each reaction contained 1 μl of cDNA template, 5 μl of TaqMan fast advanced master mix, 3.5 μl of diethylpyrocarbonate-treated water, and 0.5 μl of either of the following Taqman assays: ribosomal protein S18 Hs01375212_g1 and IL-6: Hs00174131_m1. Ct values were normalised to the reference gene ribosomal protein S18 and data analysed using the comparative ΔCt method (41 (link)).