Color optitaq pcr master mix 2

Total amount of RNA was isolated using RNeasy Plus Mini kit (Qiagen, Hilden, Germany) according to manufacturer's instruction. The amount of total RNA was assessed in spectrophotometric measurement (NanoDrop Technologies, Wilmington, DE, USA). cDNA synthesis was conducted with the use of oligo(dT) starters and GoScript Transcriptase (Promega GmbH, Germany) according to provided protocol. Prepared cDNA was utilised for the assessment of gene expression at the mRNA level utilising Color OptiTaq PCR Master Mix (2×) (EURx, Poland) according to manufacturer's instruction. Measurements were performed as previously described [22] . For primers used in the study PCR conditions were applied as follows: initial denaturation at 95 °C for 5 min; followed by 30 cycles: 30 s at 95 °C, 30 s at 58 °C (45 s at 55 °C for AR) and 30 s at 72 °C; and final extension at 72 °C for 10 min. Electrophoresis in the 1.5% agarose gel containing ethidium bromide was used for PCR product visualisation. Bands were normalised using GAPDH. Utilised primers' sequences are presented in Supplementary Materials (Table S1). Primers were manufactured by Sigma-Aldrich (St. Louis, MO, USA). Fluorescence signal of ethidium bromide was detected by Bio-Rad Chemi-Doc™ XRS + System (Bio-Rad, Hercules, CA, USA).