Qrt pcr mix

Gene expression analyses were performed by qRT-PCR. C. albicans strains were grown overnight in liquid YPD at 30 °C. Strains were then cultured in YPD at 30 °C (200 rpm) for 4 h. Cells were pelleted and washed three times with sterile PBS. Cell pellets were stored at −80 °C until processed for RNA isolation. RNA was isolated from Candida cells using the RiboPure Yeast RNA isolation kit (Ambion). Total RNA obtained after the elution step was subjected to DNase treatment using Turbo DNaseI. RNA concentration and quality was determined using a Nanodrop. One microgram total RNA was converted to cDNA using the iScript kit (Biorad). cDNA was diluted twofold and 1 μL was used for each PCR with the Biorad qRT PCR mix. Primers used for the expression analysis of NRG1, CUP9, MCM1 and PMA1 are listed in SI Appendix, Table S2. PMA1 primers were used for normalization and ddCT was determined using the parental SC5314 strain (CA10) as control. Experiments were performed with three biological replicates.

Chr 7 copy numbers were determined by quantitative real-time (qRT) PCR. In brief, the gDNA was isolated from cells grown in YPD overnight at 30 °C. The YeaStar genomic DNA kit (Zymo Research) was used for gDNA isolation. gDNA samples were diluted to 2 ng/μL to set up qRT-PCR. PCR of C. albicans Chr 1 and Chr 4 with corresponding primers was used as copy number controls (SI Appendix, Table S2). Three different primer pairs spanning different regions of Chr 7 were used for the determination of Chr 7 copy numbers (SI Appendix, Fig. S6A and Table S2). Additionally, two different sets of NRG1-specific primers were used to quantify NRG1 gene dosage (SI Appendix, Table S2). The following PCR conditions were used: 95 °C for 5 min, 95 °C for 5 s, 60 °C for 30 s, steps 2 to 3 repeated for 39 cycles, 65 °C for 31 s, 65 °C for 5 s, and ramp 0.5 °C/s. A Biorad qRT-PCR mix was used to set up reactions that were run on a Biorad CFX Maestro 384-well machine and analyzed with the Biorad CFX Maestro software.