Probe qpcr master mix

The relative expression of genes associated with adhesion and hyphal growth of, i.e., ALS1, ALS3, BCR1, CPH1, ECE1, EFG1, HWP1, HYR1, and SAP1 was evaluated using two-step real-time PCR after treatment with 4-AN (1 µg/mL). Control cells were treated with DMSO (0.1%). Total RNA was converted to cDNA using smart a First Strand cDNA Synthesis Kit (EurX, Gdańsk, Poland). For PCR detection of transcripts, we used TaqMan Gene Expression Assays (Lot: 170255, designed by manufacturer, Thermo Fisher Scientific, Waltham, MA, USA) and Probe qPCR Master Mix (EurX). The cDNA samples were pre-treated at 50 °C for 2 min with UNG to degrade any dUMP-containing PCR products and then subjected to initial denaturation at 95 °C for 10 min., followed by 40 amplification cycles with denaturation at 94 °C for 15 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s using RotorGene-6000 (Corbett Life Science, Mortlake, Australia). The relative gene-expression level of the tested genes was calculated with the 2^−(ΔΔCt) method using ACT as a reference gene. The experiment was done in triplicate.

The expression of the SAP4 gene was evaluated using real-time PCR after treatment with silymarin. The cells of C. albicans ATCC 10231 were exposed to silymarin at the concentration of 15 µg/mL (MIC/2) or 1% DMSO (control) during propagation in liquid culture in Spider medium (1% nutrient broth, 1% mannitol, 0.2% K₂PO₄ (Sigma-Aldrich), pH 7.2) with 10% fetal bovine serum (FBS) (Sigma-Aldrich). After 20-h incubation at 37 °C, the yeasts were collected and total RNA was extracted with the YeaStar RNA kit (Zymo Research, USA), according to the manufacturer’s instructions. cDNA was then synthesized using a Smart First Strand cDNA Synthesis Kit (EurX, Poland) following the manufacturer’s instructions. For PCR detection of transcripts, we used TaqMan gene expression assays (Lot: 170255, designed by manufacturer, Thermo Fisher Scientific, England) and the Probe qPCR Master Mix (EurX, Poland). The cDNA samples were pre-treated at 50 °C for 2 min with uracil-N-glycosylase to degrade any dUMP-containing PCR products and then subjected to initial denaturation at 95 °C for 10 min., followed by 40 amplification cycles with denaturation at 94 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s using RotorGene-6000 (Corbett). The relative level of expression of the tested gene was calculated with the 2^-(ΔΔCt) method using ACT1 as a reference gene [38 (link)].