Icycler iq real time detection system software

Macrophages (± Ev, Ev fractions, T. cruzi, or inhibitors) were snap-frozen in liquid nitrogen and homogenized in Trizol reagent (Invitrogen, weight/volume ratio, 1:10). Total RNA was extracted and precipitated by chloroform/isopropanol/ethanol method, treated with DNase I (Ambion, Austin, TX) to remove contaminating DNA, and assessed by spectrophotometry for purity (OD₂₆₀/OD₂₈₀ ratio > 1.8) and amount (OD₂₆₀ of 1 = 40 μg/mL) [42 (link)]. First strand cDNA was synthesized by using 2 μg total RNA and iScript^™ cDNA synthesis kit (170–8891, Bio-Rad), and stored in 100-μL nuclease free dH₂O. Real time qPCR was performed in a 20 μL reaction containing 2 μL cDNA, 10 μL SYBR green master mix (170–8882; Bio- Rad), and 20 μM of the gene-specific oligonucleotides listed in S1 Table. The thermal cycling conditions were 95°C for 3 min and 40 cycles of 95°C for 15 sec and 60°C for 30 sec. The PCR base line subtracted curve fit mode was applied for determining the threshold cycle (Ct) by using iCycler iQ real-time detection system software (Bio-Rad). For each target gene, Ct values were normalized to the mean Ct value for murine Gapdh reference cDNA. The relative expression level of target gene was calculated by following the 2^−ΔCt [2 ^{(-ΔCt sample)} / 2 ^{(– ΔCt of control)}] method [43 (link)].