Quantitative RT-PCR

Total RNA was isolated from neutrophil cultures or from the spleens and livers of WT and TLR2^−/− mice at the 4th wpi and uninfected using an SV Total RNA Isolation System Kit (Promega, Madison, WI, USA). Gene expression was normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT) expression for spleen and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression for liver and neutrophil culture. The primer sequences used were as follows: HPRT forward primer, 5′-TGGAAAAGCCAAATACAAAGC-3′, and reverse primer, 5′-CAACATCAACAGGACTCCTCG-3′; GAPDH forward primer, 5′-TGCAGTGGCAAAGTGGAGAT-3′, and reverse primer, 5′-CGTGAGTGGAGTCATACTGGAA-3′; TLR2 forward primer, 5′-AAGTCTCCGGAATTATCAGTCC-3′, and reverse primer, 5′-TGATGGATGTCGCGGAT-3′; and iNOS forward primer, 5′-CGAAACGCTTCACTTCCAA-3′, and reverse primer, 3′-TGAGCCTATATTGCTGTGGCT-5′.