Quantitative RT-PCR Analysis of Immune Markers

qRT-PCR analysis was performed on an ABI step one plus real-time system (Applied Biosystems). Total cellular RNA was isolated from each sample by using a Qiagen RNeasy Kit (Qiagen, Valencia, CA). One microgram of total RNA from each sample was subjected to cDNA synthesis using the Superscript III reverse transcriptase (Invitrogen). cDNAs were amplified by PCR with primers as follows: Perforin (sense, 5′-TCCTATGGCACGCACTT TATCAC-3′; antisense, 5′-TCCACGTTCAGGCAGTCTCCTAC-3′); Granzyme B (sense, 5′-GCTGCTAAAGCTGAAGAGTAAGG-3′; antisense, 5′-GCGTGTTTGAGTATTTGCCC A TT-3′); TGF-β (sense, 5'-TGGAAACCCACAACGAAATCT-3′; antisense, 5'-GCTGAGGT ATCGCCAGGAAT-3′); β-actin (sense, 5′-TTTCCAGCCTTCCTT CTTGGGTAT-3′; antisense, 5′-TGTTGG CATAGAGGTCTTTACGG-3′). The mRNA levels of the genes of interest were expressed as the ratio of each gene of interest to β-actin for each sample. SYBR Green quantitative PCR amplifications was performed in the Step one plus Detection System (Applied Biosystems). The comparative Ct (ΔΔCt) method was used to determine the expression fold change [3 (link)].