Rnaqueous micro kit column purification

CD8⁺ T cells or CD11b⁺ myeloid cells were enriched from tumor-cell mixtures, TDLNs or spleens from B16 tumor-bearing Stat3^+/+ or Stat3^−/− mice, as described above. In some experiments, CD11b⁺F4/80⁺ myeloid cells were sorted by FACSAria SORP cell sorter (BD Bioscience), from tumor-cell mixtures. Total RNA was extracted using RNAqueous Micro kit column purification (Ambion). cDNA was produced from RNA using cDNA synthesis kit (Bio-Rad). Quantitative RT-PCR was performed using SYBR Green Supermix (Bio-Rad) and quantified using Chromo4 real-time detector (Bio-Rad). RT-PCR primers were purchased from SA Biosciences. Each primer set was validated using a standard curve across the dynamic range of interest with a single melting peak.

Total RNA was extracted using RNAqueous Micro kit column purification (Ambion). cDNA was produced from RNA using cDNA synthesis kit (Bio-Rad). Quantitative RT-PCR was performed using SYBR Green Supermix (Bio-Rad) and quantified using Chromo4 real-time detector (Bio-Rad). RT-PCR primers were purchased from RealTimePrimers.com. Each primer set was validated using a standard curve across the dynamic range of interest with a single melting peak.