Dna oligonucleotides

Total RNA was isolated with different promastigote dilutions using trizol reagent (Roche) as performed previously (19). The quantity of the extracted RNAs was analysed using a NanoDrop spectrometer 100 ng RNA was added per reaction. Synthesis of cDNA was performed using a cDNA Synthesis Kit (EvoScript Universal cDNA Master, Roche) with random hexamer primers according to the protocol provided by the manufacturer (15 min at 42 °C, 5 min 85 °C, 15 min at 65 °C and cool to 4 °C with an unlimited hold time). The modification was done prior to cDNA synthesis, the samples were denatured at 95 °C for 2 min, as a modification of the kit procedure. qPCR Quantitative real-time PCR was performed with a reaction solution of 0.5 mM primers in SYBR Green Master mix (Roche). The reaction performed with an initial denaturation (95 °C for 5 min) followed by 40 amplification cycles (10 s at 95 °C, 10 s at 55 °C, 10 s at 72 °C) and a fluorescence detection step (70 °C to 95 °C) with quantification of amplified DNA after each cycle. The DNA oligonucleotides (Microsynth, Switzerland) targeted LRV 2-1 RNA Dependent RNA polymerase (RdRp) gene were used (18,19). Molecular grade water was used as negative control.