At regular time intervals, fluorescence was measured using a microtiter plate reader (Wallac Victor; Perkin Elmer). The excitation and emission wavelengths for 4-MU are 355 nm and 460 nm, respectively.
Linear regression analysis was performed on the data and the relative slopes were calculated from the fluorescence-time curves of start cultures and biofilms as follows: (slope of the curve for biofilms/slope of the curve for start AZD8931 cultures)*100. Data were obtained in three independent experiments. One-way ANOVA tests were carried out using SPSS 15.0 software to determine whether differences were statistically significant (p < 0.05). Real-time PCR Biofilms and start cultures were grown and harvested as described above. Samples were obtained from at least four independently-grown biofilms (n ≥ 4) and from six independently-grown start cultures (n = 6). RNA extraction and cDNA synthesis were Liver X Receptor agonist performed as described previously [20]. Primers for the ALS genes were obtained from the study of Green et al. [47], and primers for the other genes and for the reference genes were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA). Full-length gene sequences were obtained from the C. albicans database http://www.candidagenome.org/[48]. The specificity of each primer was checked by comparing its sequence to the C. albicans database using BLAST
[49]. The sequences of the primers developed in the present study are given in Table 1 and Table 2, and for all the primers a concentration of 300 nM was used (except for PMA1 for which 600 mafosfamide nM was used). For SAP7 and SAP8, no good quality primers were obtained, and therefore these two genes were selleck chemical excluded from the present study. Real-time PCR was performed in 96-well plates using the ABI PRISM® 7000 apparatus (Applied Biosystems) and the MESA GREEN qPCR masterMix Plus for SYBR Assay I dTTP kit (Eurogentec, Seraing, Belgium). Five μl of 1:2 diluted cDNA samples and 20 μl of mastermix (containing the primers) were added to the plates. Real-time PCR reactions were performed at 95°C for 5 min, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Following PCR, samples were subjected
to incubations of increasing temperature starting from 60°C to 95°C to obtain melt curves. Samples were also subjected to gelelectrophoresis as described previously [20]. Control samples were included on each plate to ensure that multiple plates could be compared. Control reactions were also performed with RNA that had not been reverse transcribed in order to ensure that no genomic DNA was amplified during the PCR reactions. Real-time PCR data were normalized with the geometric mean of five reference genes. ACT1, RIP, RPP2B, PMA1 and LSC2 were used for this purpose, as they have previously shown to be stably expressed in C. albicans biofilms and planktonic cells [20]. Normalized data were then used to calculate the relative gene expression levels.