Quantitect reverse transcription kit protocol

Complementary DNA (cDNA) was prepared according to Quantitect reverse transcription kit protocol (Qiagen, Hilden, Germany). The concentration and purity of the resultant cDNA were evaluated using UV-Visible Spectro Nanophotometer. The prepared cDNA was diluted ten times, concentrations were measured again, and then the diluted cDNA was stored at −20 °C. Two negative controls were included a no template control (NTC), a full reaction without RNA template to serve as a general control for extraneous nucleic acid contamination. The second control was a no reverse transcriptase control (NRT) or minus reverse transcriptase control (-RT).

Total RNA was isolated from yeast wild type strain using RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol and transcribed into cDNA according to the QuantiTect reverse transcription kit protocol (Qiagen). For quantitative real-time PCR 20 μl triplicate reactions with 1 μl of 1:5 diluted cDNA were used together with 0.2 mM of the primer pair and 1x GoTaq®qPCR Master Mix (Promega). For detection the ABI 7500 Fast Real-Time PCR System (Applied Biosystems) was used. Data were analyzed using the comparative 2ΔΔCT method and TAF10 as a reference gene. Experiments were repeated at least three times.