mRNA expression analysis
Quantitative real time PCR (qPCR) was performed to estimate mRNA level expression of IDO, KAT II, KMO, PNPO enzymes. HPRT (hypoxanthine phosphoribosyl-transferase) was used as house-keeping gene. All experiments were performed in triplicates. Specified primers for all the genes were designed using Primer3 and BLAST (Primer3 Input version 0.4.0 and BLAST; http://www.ncbi.nlm.nih.gov/tools/primer-blast/). For each target gene, primers were designed to target all possible transcript variants mentioned in the reference sequence (RefSeq) database (http://www.ncbi.nlm.nih.gov/refseq). RNAs were extracted using Trizol reagent (Qiagen RNeasy kit); quantified using Qubit 3 fluorometer (Thermo scientific) and Qubit RNA BR assay kit. Reverse transcription was performed to create cDNA using high capacity cDNA reverse transcription kit (Thermo scientific) according to the manufacturer’s protocol. qPCR was performed with cDNA, suitable primers using SsoFast Eva Green Supermix (Bio-Rad) on a CFX-96 real time system (Bio-Rad). The cycle parameter was: 5mins at 95ºC for initial hot-start, followed by 40 cycles of 15secs at 95ºC for denaturation and 30secs at 60ºC for annealing and extension. Specificity of the expected PCR products were verified using melting-curve analysis. CTvalues (cycle threshold) were obtained for individual samples and delta CT (ΔCT) were calculated by subtracting the CT values of house-keeping gene (HPRT) from that of the gene of interests. These ΔCT values were used to compare extent of the mRNA expression between the two groups where mRNA expression was inversely proportional to the ΔCT value (Livak & Schmittgen, 2001). The primer sequences were: for HPRT-F, 5’-GCTTTCCTTGGTCAGGCAGTA-3’, for HPRT-R, 5’-GGTCCTTTTCACCAGCAAGCT-3’, for IDO-F, 5’-GCGCTGTTGGAAATAGCTTC-3’, for IDO-R, 5’-TGATCGTGGATTTGGTGAAA-3’, for KATII-F, 5’-TTAATGCTCCCTGGAAATGC-3’, for KATII-R, 5’-GAAGGCCACATCCATCTGTT-3’, for KMO-F, 5’-CACTGTGTACTGCTGGGAGA-3’, for KMO-R, 5’-ATCGCGTGATCATCTGGGAT-3’, for PNPO-F, 5’-ACCCAGGCCCTTCTTTCTAA-3’, and for PNPO-R, 5’-TACGCCACCATTACACTCCA-3’.