4.3 (c) Expression profile of downstream effectors- MyD88 and NF-ƙB using RT-PCR
mRNA was isolated from unstimulated and either Rv2615c (10μg/ml) or LPS (40ng/ml) stimulated THP-1 macrophages after 16h, 24h and 48h using mRNA synthesis kit (Promega Corporation) as per manufactures’ protocol. 1µg of mRNA was converted to cDNA (Promega Corporation) using cDNA synthesis kit. mRNA and cDNA were checked by setting up PCR of endogenous control i.e., housekeeping gene GAPDH. Quantitative real time PCR for downstream effector genes- MyD88 and NF-ƙB was performed by relative quantification in which the target concentration is expressed as ratio of target vs. reference gene (housekeeping gene GAPDH). The expression of MyD88 was evaluated following 24h of protein stimulation and the expression of NF-ƙB was evaluated following 24h and 48h of protein stimulation. The primers used were Forward Primer 5’ ATG GCT TCT ATG AGG CTG AG 3’ and Reverse Primer 5’ GTT GTTGTT GGT CTG GAT GC 3’. Equal concentration of cDNA for each sample was added to SYBR PCR master mix along with respective gene primers. 40 cycles of amplification followed by data acquisition and analysis was done. Data was calculated using the 2-∆∆ CT method and are presented as fold induction. Fold change was normalized to GAPDH expression levels. Experiments were repeated thrice.