Rt pcr mix sybr

The total RNA was extracted using RNA extracol (EurX, Gdansk, Poland) according to the manufacturer’s instructions. RNA quality was checked on the 1% agarose gel and the quantity of RNA on the NanoDrop One/One UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Next, 1 µg of RNA was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the producer’s instructions and qPCR was performed using the RT PCR Mix SYBR^® (A&A Biotechnology, Gdańsk, Poland) on the CFX Opus Real-Time PCR Systems (Bio-Rad, Hercules, California, USA). The following primers were used: target gene TYMS (Fw 5′-TGGAATCCAAGAGATCTTCCTC-3′; Rv 5′-TTCAGGCCCGTGATGTG-3′), target gene DHFR (Fw 5′-GCGTTCTGCTGTAACGAG-3′; Rv 5′-ACCAGATTCTGTTTACCTTCTAC-3′), target gene CSNK2A1 (Fw 5′-GGTGAGGATAGCCAAGGTTCTG-3′; Rv 5′-TCACTGTGGACAAAGCGTTCCC-3′) and reference gene GAPDH (Fw 5′-AGGGCTGCTTTTAACTCTGGT-3′; Rv 5′-CCCCACTTGATTTTGGAGGGA-3′). The PCR cycling conditions were set as follows: initial denaturation for 3 min at 95 °C, and then 40 cycles of 15 s at 95 °C, 60 s at 55 °C and 45 s at 72 °C. The post-PCR melting curve was obtained using 15 s at 95 C, 1 min at 60 C, and 15 s at 95 C. The relative fold gene expression was calculated using the 2^−ΔCt method [49 (link)], and the results were expressed as a percent of control.