Real-time quantitative PCR (Applied Biosystems, USA) was performed to verify the mRNA expression in the trigeminal ganglia and brain of mice infected with HSV-1. The reactions were performed using the Sybr Green PCR Master Mix (Applied Biosystems, USA) in the Applied Biosystems’ 7000 Sequence Detection System, at 50°C, 2’; 95°C, 10’; and 40 cycles of 95°C, 15” and 60°C, 1’, followed by a final dissociation stage. The oligonucleotides used in the reactions were specific for HPRT,
F - GTT GGA TAC AGG CCA GAC TTT GTT G and
R - GAT TCA ACT TGC GCT CAT CTT AGG C; IP-10 (CXCL10),
F -GCC GTC ATT TTC TGC CTC AT and
R - GCT TCC CTA TGG CCC TCA TT; TNF alpha,
F - CAT CTT CTC AAA ATT CGA GTG ACA A and
R - TGG GAG TAG ACA AGG TAC AAC CC; iNOS,
F - CAG CTG GGC TGT ACA AAC CTT and
R - CAT TGG AAG TGA AGC GTT TCG; MCP-1(CCL2),
F - CTT CTG GGC CTG CTG TTC A and
R - CCA GCC TAC TCA TTG GGA TCA [
36]; RANTES (CCL5),
F - GTC GTG TTT GTC ACT CGA AGG A and
R - GAT GTA TTC TTG AAC CCA CTT CTT CTC; gp91,
F - CCA ACT GGG ATA ACG AGT TCA AGA C and
R - AAG GCT TCA GGG CCA CAC A; p22,
F - TGG CTA CTG CTG GAC GTT TCA C and
R - CTC CAG CAG ACA GAT GAG CAC AC; VP16
F - TTT GAC CCG CGA GAT CCT AT and
R - GCT CCG TTG ACG AAC ATG AA [
37]; TLR1,
F - TGA TCT TGT GCC ACC CAA CA and
R - GCA GGG CAT CAA AGG CAT TA; TLR2,
F - TTG CTC CTG CGA ACT CCT AT and
R - AGC CTG GTG ACA TTC CAA GA; TLR3,
F - TAG ACT GCA TCG CCT GCT AA and
R - AAG CAG CCA GAA GCA GAA CT; TLR6,
F - TCT GGG ATA GCC TCT GCA ACA and
R - GGC GCA AAC AAA GTG GAA AC; TLR7,
F - TAC CAG GAC AGC CAG TTC TA and
R - AGG AGC CTC TGA TGA GAC AA; and TLR9,
F - ACC TCA GCC ACA ACA TTC TC and
R - TGC ACC TCC AAC AGT AAG TC [
38]. The comparative Ct method was chosen to analyze the data, using the arithmetic formula 2
-ΔΔCt[
39]. The genes expression was normalized to expression of the constitutively expressed gene Hypoxanthine-guanine phosphoribosyltransferase (HPRT). All of the reactions were replicated.