Quantitative PCR Analysis of Immune Genes

Total RNA was extracted with RNeasy Mini Kit (Qiagen). The amount of 1 μg of RNA was reverse transcribed to cDNA using random hexamer primers from GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster City, CA) in a 20 μL reaction volume at 42^oC for 30 min. Quantification of PCR products was performed in 10 μL of Lightcycler 480 SYBR Green I Master mix (Roche) using a real-time PCR Lightcycler (Roche). DNA was denatured at 95^oC for 2 min; then followed 45 cycles of denaturation at 95^oC for 25 s, annealing at 60^oC for 45 s, elongation at 72^oC for 1 min and incubation at 80^oC for 5 s. cDNAs were amplified with specific primers for β-actin, TAP-1, LMP-2, TAP-2, and LMP-7. The list of the TAP-1, TAP-2, LMP-2, LMP-7 and reference genes and their primer sequences have been described elsewhere [15 (link),18 (link)]. Fold changes in the transcript levels were calculated using C_T values standardized to β-actin, used as the endogenous reference gene control. All samples were run in biological triplicates. For statistical analysis of qPCR the Student's t-test was used. Differences between experimental and control samples with P< 0.05 were considered to be statistically significant. The levels of relative gene expression were presented as fold changes compared to the levels found in control samples.