9. Gene expression analyses
Gene expression was analyzed by quantitative real-time PCR performed in Rotor-Gene Q cycler (Qiagen) with QuantiNova SYBER® Green PCR Master Mix 2X (Qiagen). All PCR reactions were performed in triplicate in a total volume of 20 µL for 40 cycles under the following conditions: initial activation, 95°C, 2 min; 40 cycles of denaturation, 95°C for 5 sec and combined annealing/extension, 60°C, 10 sec. Threshold cycles (Ct) were calculated using the β-tubulin gene as endogenous control, which was selected due to its expression stability among other housekeeping genes, such as actin and tef-1 . Data were expressed as 2-ΔΔCt (Livak & Schmittgen, 2001). Values from three biological replicates were consistent and used for ANOVA statistical analysis, using SYSTAT©v.13.2 software. Data from liquid cultures ofTgam was analysed by ANOVA and Tukey test. Primers used for assessing expression patterns of ts1 , ts3 , ts4 ,ts5 , ts6 , ts7 , ts9 , ts11 , tri5genes and those of β-tubulin (Supporting Information TableS3 ) were checked for efficiency and dimmer formation.