RNA Extraction and qRT-PCR Analysis

RNA was isolated and purified using Total RNA Isolation kit with mini column system (A&A Biotechnology, Gdynia, Poland). Concentration and purity of RNA samples were determined using a NanoQuant plate reader (Infinite M200Pro, Tecan). Total RNA (1 μg) was transcribed into cDNA using 300 ng of random primers and SuperScript II Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, CA, USA). The evaluation of mRNA expression of selected genes was performed by quantitative RT-PCR using the Rotor-Gene 3000 Real-Time DNA analysis system (Corbett Research, Morklake, Australia). Amplification was performed using KAPA SYBR FAST qPCR Kit Universal 2X qPCR Master Mix (Kapa Biosystems, Cape Town, South Africa), 200 nM of each primer and 25 ng cDNA template per reaction. Sequences of primers used in qRT-PCR were shown elsewhere [8 (link),21 (link)]. To calculate the relative expression of target genes versus a reference gene RPS17, a mathematical model including an efficiency correction was used.