Gene expression changes were monitored as previously described [31 (link)]. Briefly, strains were subcultured to mid-log phase, pelleted at 3,000 rpm at 4°C, washed once, and snap frozen. Cells were lysed by bead beating, RNA was extracted using the QIAGEN RNeasy kit, and DNase treated using the QIAGEN RNase free DNAase Set, and cDNA was amplified by using the AffinityScript Multi Temperature cDNA Synthesis Kit (Agilent Technologies, Mississauga, ON, Canada) with the provided random primers.
qRT-PCR was performed using a 384-well plate, with a 10 μL reaction volume using Fast SYBR Green Master Mix (Applied Biosystems) and the BioRad CFX-384 Real Time System with the following cycling conditions: 95°C for 3 minutes, then 95°C for 10 seconds and 60°C for 30 seconds, for 40 cycles. The melt curve was completed with the following cycle conditions: 95°C for 10 seconds and 65°C for 10 seconds, with an increase of 0.5°C per cycle up to 95°C. Reactions were performed in triplicate for two biological replicates. All data were normalized to the PMA1 and TEF1 reference genes for C. albicans. Additional gene primer pairs are included inS7 Table . Data were analyzed using the BioRad CFX Manager 3.1. Error bars depict standard error of the means of technical triplicates.
qRT-PCR was performed using a 384-well plate, with a 10 μL reaction volume using Fast SYBR Green Master Mix (Applied Biosystems) and the BioRad CFX-384 Real Time System with the following cycling conditions: 95°C for 3 minutes, then 95°C for 10 seconds and 60°C for 30 seconds, for 40 cycles. The melt curve was completed with the following cycle conditions: 95°C for 10 seconds and 65°C for 10 seconds, with an increase of 0.5°C per cycle up to 95°C. Reactions were performed in triplicate for two biological replicates. All data were normalized to the PMA1 and TEF1 reference genes for C. albicans. Additional gene primer pairs are included in