Quantitative PCR Validation of aCGH Data

qPCR was used to validate the aCGH data. Primer amplification efficiency for each gene was determined. Specific sequences of the reference and target genes were cloned into pGEM-T easy vector (Invitrogen). Known amounts of genomic DNA (from clones D11 and CLB and G strain) and recombinant plasmids containing the sequences of interest were incubated with 10 µL SYBR Green-Based Detection (Applied Biosystems), sense and antisense primers and water to a total reaction volume of 20 µL. The reaction mixture was distributed into 0.2 mL tubes and subjected to 40 cycles of amplification in the Rotor-Gene^® Q PCR cycler (Qiagen) according to the manufacturer’s instructions. The qPCR program was set as follows: initial denaturation at 95°C for 5 min, 40 cycles of denaturation at 95°C for 15 s, annealing and extension at 60°C for 60 s. The results were analyzed with Rotor-Gene 6000 v1.7 software (Qiagen). A standard curve was constructed for each target gene. Data obtained by amplification with genomic DNA samples could be compared as the amount of genomic DNA was the same for the three T. cruzi isolates. To estimate the copy numbers of each target gene in the three isolates, data were normalized separately using the genome size of each T. cruzi isolate (Souza et al., 2011 (link)). All experiments were performed in triplicate.