albicans from blastospore to hyphal form, the culture medium PF-6463922 was supplemented with 10% fetal calf serum and the incubation was performed for 3 and 6 h at 37°C. Following each culture period under both conditions , the cultures were centrifuged 10 min at 13,000 rpm, the supernatants were discarded, and each pellet was suspended thereafter in 0.6 ml of lysis buffer (Glycerol 1 M, EDTA 0.1 M). Glass beads (0.425-0.6 mm in diameter; 0.2 ml) were added to each suspended pellet prior
to sonication (4 × 1 min, followed by 2 min of incubation in ice) with a MiniBead-beater (Biospec Products, Bartlesville, OK, USA). Following cell lysis, the total RNA was extracted from each sample by means of the Illustra RNAspin Mini kit (GE Health Care UK Limited, Buckingham, UK). Concentration, purity, and quality of the isolated RNA were determined using the Experion system and RNA StdSens analysis kit according to the instructions provided by the manufacturer (Bio-Rad, Hercules, CA, USA). Quantitative real-time RT-PCR The RNA (500 ng of each sample) was reverse transcribed into cDNA by means of the iScript cDNA Synthesis kit (Bio-Rad, Mississauga, ON, Canada). The conditions
for the preparation of the cDNA templates for PCR analysis were 5 min at 25°C, 1 h at 42°C, and 5 min at 85°C. Quantitative PCR (qPCR) was carried out as previously described . The quantity of mRNA transcripts was measured with the Bio-Rad CFX96 real-time PCR detection system. Reactions were performed selleck screening library using a PCR supermix, also from Bio-Rad (iQ SYBR Green supermix). Primers (Table 6) were added to the CB-5083 cell line reaction mix to a final concentration of 250 nM. Five microliters of each cDNA sample were added to a 20 μl PCR mixture containing 12.5 μl of the iQ SYBR Green supermix, 0.5 μl of specific primers ACT1, SAP2, SAP4, SAP5, SAP6, HWP1, and EAP1 (Midland Certified Reagent Company, Inc., Midland, TX, USA), as well as EFG1 and NRG1 (Invitrogen Life Technologies Inc., Burlington, ON, Canada), and 7 μl of RNase/DNase-free
water (MP Biomedicals, Solon, OH, USA). Each reaction was performed in a Bio-Rad MyCycler Thermal Cycler. For the qPCR, the CT was automatically determined using the accompanying Bio-Rad CFX Manager. The thermocycling Thalidomide conditions for the ACT1, SAPs 2-4-5-6, and EAP1 were established as 5 min at 95°C, followed by 30 cycles of 15 s at 95°C, 30 s at 60°C, and 30 s at 72°C, with each reaction performed in triplicate. For the EFG1 and NRG1, the thermocycling conditions were set for 3 min at 95°C, followed by 45 cycles of 10 s at 95°C, 40 s at 54°C, and 40 s at 72°C, with each reaction also performed in triplicate. For the HWP1, the conditions were 3 min at 95°C, followed by 45 cycles of 10 s at 95°C, 30 s at 54°C, and 40 s at 72°C, with each reaction performed in triplicate. The specificity of each primer pair was determined by the presence of a single melting temperature peak.