The expressions of COX-2, MIP1β and TNFα transcripts were determined with real-time PCR performed on Light cycler 480 (Roche). Cells were cultured and treated as described above, and total RNA was isolated with an RNAeasy minikit (Qiagen) according to manufacturer’s instructions. RNA concentration was determined using a Take 3 module of Epoch micro plate reader (Biotek) at 260/280 nm. 1µg of total RNA was used for reverse transcription using cDNA synthesis kit from Quanta Biosciences, containing MgCl2, dNTPs, recombinant RNAse inhibitor protein, qScript Reverse Transcriptase, random primers, oligo (dT) primers and stabilizers. Gene expression was assayed by quantitative real-time PCR on LightCycler® 480 II (Roche Applied Science) using LightCycler® 480 SYBR Green I Master mix, cDNA prepared as described above and COX-2, MIP1β, TNFα and GAPDH forward and reverse Primers. Following are the forward (F) and reverse (R) primers used
MIP1β–F- CCAGCCAGCTGTGGTATTR-CAGTTCAGTTCCAGGTCATACATNFα-F- CCAGGGACCTCTCTCTAATCAR-TCAGCTTGAGGGTTTGCTACCOX-2-F-CAACTCTATATTGCTGGAACATGGAR-TGGAAGCCTGTGATACTTTCTGTACTGAPDH-F-TGCACCACCAACTGCTTAGCR-GGCATGGACTGTGGTCATGAGThe ΔΔCt values for COX-2, MIP1β, and TNFα were calculated relative to the GAPDH levels and values were expressed as fold change over the control (Duah et al., 2013 (link)).