RT-qPCR analysis of gene expression

RT-qPCR analyses were carried out with SYBR Green I protocol on PikoReal 96 (Thermo Fisher) [42 (link)]. A plate layout with duplicates for each cDNA and primer was created in advance. Suitable sequences of nucleotides were searched in PubMed and primers were blasted with PubMed tool. On 96-well plates a final volume of 20 µL/well (5 µL cDNA mix and 15 µL Primermix, Thermo Fisher) was applied. Every analyzed gene was normalized to the housekeeping genes β-ACTIN (ACTB) and Histocompatibility 3 (H3; Table 2). Ct-values of each sample were analyzed with PikoReal 2.2 software (Thermo Fisher). By using REST software (Qiagen, Hilden, Germany), significances between stressed groups and the control group were evaluated.