Fast probe qpcr master mix

In the experiment, the relative expression of the Tnfrsf1a and Cav1 genes was measured, using Hprt and Tbp as endogenous controls (Table 1). The reaction was performed in triplicate using the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and Fast Probe qPCR Master Mix (2x) plus ROX solution (EURx, Gdańsk, Poland). The reaction mixture contained 10 µL of Fast Probe qPCR Master Mix (2x), 9 µL of RNase-free water, ROX solution (50 nM), and 0.5 µM of the gene-specific TaqMan probe (Applied Biosystems, Foster City, CA, USA). Data quality screening was performed based on amplification, Tm and Ct values to remove any outliers before calculating ΔΔCt and determining the fold change in mRNA levels. The data is presented as an RQ value (RQ = 2 − ΔΔCt).
The primer sequences, gene symbols, assay IDs, gene names, and amplicon lengths (bp) for the genes analyzed are presented in Table 1.

The relative expression of the following genes was measured: Slc2a3, Gapdh, Ldha, Ldhb, and Pkfb3 by real-time PCR, ΔΔCt, using Hprt and Tbp as endogenous controls (Table 1). The reaction was performed in successive triplicates using a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and Fast Probe qPCR Master Mix (2×) plus ROX solution (EURx, Gdańsk, Poland). The reaction mixture contained 10 µL of Fast Probe qPCR Master Mix (2×), 9 µL of RNase-free water, ROX solution (50 nM), and 0.5 µM of the gene-specific TaqMan probe (Applied Biosystems, Foster City, CA, USA). Data quality screening was performed based on amplification, Tm, and Ct values to remove any outliers before calculating ΔΔCt, and determining the fold change in mRNA levels. Data are presented as RQ value (RQ = 2 − ΔΔCt).

The expression of HWP1, SAP4, ALS1, ALS3, HYR1, BCR1, EFG1, ECE1, and CPH1 genes was evaluated using real-time PCR after treatment with quinalizarin. The C. albicans cells were exposed to anthraquinone at the concentration of MIC/2 (4 µg/mL) or 1% DMSO (control) during propagation in liquid culture in Spider medium with 10% fetal bovine serum. After 10-h incubation at 37°C, the yeast cells 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 the manufacturer, ThermoFisher Scientific, England) and the Fast Probe qPCR Master Mix (EurX, Poland). The cDNA samples were pre-treated with uracil-N-glycosylase at 37°C for 2 min to degrade any dUMP-containing PCR products and then subjected to initial denaturation at 95°C for 3 min, followed by 40 amplification cycles with denaturation at 95°C for 10 s and annealing/extension at 60°C for 30 s using QuantStudio3 (Applied Biosystems). The relative level of expression of the analyzed genes was calculated with the 2-(∆∆Ct) method using ACT1 as a reference gene (43 (link)).