Total RNA from biofilms was isolated using the RiboPure yeast kit (Ambion, Inc.), according to the manufacturer’s instructions. RNA concentrations and purity were determined by measuring the absorbance at 260 nm and 280 nm (ND-1000 spectrophotometer, NanoDrop Technologies). Equal amounts of RNA (3 μg in 20 μl reactions) were reverse transcribed with oligo(dT) primers using Superscript Anlotinib reverse transcriptase II (Invitrogen). Primers were based on the published sequence of the EFB1 gene of C. albicans. Primer sequences used were as follows: Forward:

5′- CAT TGA TGG TAC TAC TGC CAC -3′; Reverse: 5′- TTT ACC GGC TGG CAA GTC TT -3′. The forward primer spanned the sole exon-exon boundary of EFB1, thus excluding amplification of genomic C. albicans DNA. The uniqueness of the primers for C. albicans EFB1 was determined Selleckchem DihydrotestosteroneDHT using the BLAST database http://​www.​tigr.​org. To generate standard curves for quantitative analyses a pEFB plasmid was prepared as follows. A 136-bp C. albicans EFB exon fragment, containing the target sequence, was amplified with the above mentioned primers. PCR was ��-Nicotinamide price performed in a DNA thermal cycler with 1 cycle of 5 min at 95°C; 40 cycles of 1 min at 95°C, 30 s at 62°C, 30 s at 72°C; and a final extension at 72°C for 5 min. This fragment was ligated into the pCR 2.1 plasmid vector (3.931 kb) and transformed into One Shot cells (Top10F’) using a TA

cloning kit (Invitrogen). Plasmids were digested with xhoI to generate a linear template and purified with the PureYield™ Plasmid Miniprep System (Promega). Plasmid concentrations were determined spectrophotometrically and copy numbers calculated based on linear plasmid mass. Serial plasmid dilutions (500 pg, 50 pg, 5 pg, 500 fg, 50 fg, 5 fg, 1 fg of DNA/μl) were then used to generate standard curves for detection and quantification of EFB1 mRNA by the iCycler iQ RT-PCR assay. Real-time PCR was performed with an iCycler iQ

real-time PCR detection system (Bio-Rad). All PCR reaction mixtures contained the following: 10 μl 2 × iQ™ SYBR® Green Supermix (BioRad, Hercules, CA), 1 μl of first-strand cDNA reaction mixture or linear plasmid DNA, 0.1 μM of primers Smoothened and H2O to bring the final volume to 20 μl. The program for amplification had an incubation step at 50°C for 2 min, and 95°C incubation for 5 min, followed by 40 cycles of 95°C for 10 s and 62°C for 30 s. Reactions to estimate transcript copy number were run in duplicate from two biologic RNA replicates. Data were analyzed using the iCycle iQ system software (BioRad). Testing of planktonic cells Candida cells were grown overnight in YPD broth as described above. Cultures were adjusted to a cellular density equivalent to 1.0 × 106 cells/ml and subjected to caspofungin (CAS, Merck Research Laboratories, Rahway, N.J.) or fluconazole (FLU, Pfizer Inc.