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’.