FIG. 3.

Q-RT-PCR validation of cDNA microarray results for PBMCs from control cows. Genes encoding Sentrin-(SUMO-1), MMP 1, and MMP 23 were selected for Q-RT-PCR validation from among the genes that exhibit differential expression in nil-stimulated and M. avium subsp. paratuberculosis-stimulated PBMCs from control uninfected cows. The gene encoding Sentrin-(SUMO-1) was selected as a representative of genes that exhibit up-regulation in control cow PBMCs stimulated with M. avium subsp. paratuberculosis compared to expression in nil-stimulated cells. The genes encoding MMP 1 and MMP 23 were selected because each was apparently down-regulated by M. avium subsp. paratuberculosis stimulation of control cow PBMCs on cDNA microarrays and because of previous data suggesting that MMP gene regulation is a major and consistent effect of M. avium subsp. paratuberculosis on PBMCs from infected cows (13). For these reasons, an analysis of nil-stimulated and M. avium subsp. paratuberculosis-stimulated PBMCs from infected cows was also included in this study. Q-RT-PCR was conducted as described in Materials and Methods by using gene-specific primers. Data were analyzed by using the 2^−ΔΔCt method essentially as described previously (21) with β-actin as the control gene and nil stimulation within animal as the calibrator. The data are the means ± standard errors of the means for independent results from four infected cows and three control cows. MPTb, M. avium subsp. paratuberculosis.