Superscript 3 synthesis

A total of 10 genes were selected for qRT-PCR validation from our original 848 candidates based on gene ontology, immunogenic features, and a literature survey. All human primer sequences were designed by Integrated DNA Technologies PrimerQuest software (http://www.idtdna.com/scitools/scitools.aspx) and used at a concentration of 5μM per reaction (Supplemental Table 1). To validate the selected genes’ expression patterns, we performed qRT-PCR with SYBR Green PCR Master Mix (Applied Biosystems), on the donor cDNAs used for microarray hybridization, in addition to, cDNA derived from additional donors under the same infection conditions described above. All cDNA was generated according to manufacturer's protocols for Superscript III First Strand Synthesis System kit (Invitrogen, Carlsbad, CA). Total RNA from uninfected or Leishmania-infected DCs was isolated using an RNEasy kit (Qiagen, Valencia, CA) and 1μg of RNA per infection sample was used to generate cDNA using SuperScript III Synthesis (Invitrogen, Carlsbad, CA). For each gene, relative numbers of mRNA copies were determined by the ΔΔC_T method (10 (link)). Briefly, experimental C_T values were normalized to HPRT C_T values and depicted as fold change over uninfected samples (calibrator). The formula used is: ΔΔC_T = ΔC_{T(experimental)} - ΔC_{T(calibrator)} , where ΔC_T equals C_{T(experimental)} -C_T(HPRT).