Different biological replicate samples (Bio A to Bio G and Plankt A to Plankt F for biofilm and planktonic samples, respectively) were obtained after 24 hours of growth, as described in Materials and Methods. Total RNA was extracted from biofilms and planktonic cells (grown in three independent experiments) and cDNA synthesis was performed. The RNA and cDNA concentrations of all the samples are listed in Table 1. The average RNA and cDNA concentrations of the planktonic cells were (mean ± SD) 8.31 ± 1.5 μg/μl and 25 ± 2.61 μg/ml, respectively. The average RNA and cDNA concentrations of the biofilm cells were 4.36 ± 2.91 μg/μl and 18.83 ± 9.45 μg/ml, respectively.

Standard curves were generated for all the sample/gene combinations by using the Cycle Threshold (C_t) value versus the logarithm of each dilution factor. The linear correlation coefficient ranged from 0.9764 to 1.000. Initial amplification efficiencies (E) (obtained using 300 nM primers and 300 nM or 200 nM probe) ranged from 80% to 100%, except for the CPA1, IMH3 and LSC2 genes (E < 70%). Various primer and probe concentrations were retested for those genes, until efficiencies of >80% were obtained (data not shown). Agarose gel-electrophoresis of the amplified products showed a single band of the expected size for each qPCR assay. No primer dimers or non-specific amplification products could be observed. The inter-plate variation was < 0.5 C_t for every gene tested.

The M value of the eight housekeeping (HK) genes for all the samples (biofilm and planktonic cells) and biofilm and planktonic cells separately are listed in Table 2. This M value is defined as the average pairwise variation of one particular gene compared to all the other control genes. The gene with the lowest M value is considered as the most stable gene and the gene with the highest M value is excluded. A new M value is calculated and this procedure is repeated until only two genes are left. These two genes have the lowest M value and are therefore most stably expressed in all the samples. In Fig. 1, 2 and 3, the eight control genes are ranked according to their increasing expression stability (decreasing M value), for all samples (Fig. 1), planktonic cells (Fig. 2) and biofilm cells (Fig. 3). For all samples combined, the genes are ranked from most stable to least stable as follows: ACT1 and PMA1, RIP, RPP2B, LSC2, IMH3, CPA1 and GAPDH (Fig. 1). No significant improvement in normalization could be observed by using more than five control genes (Fig. 4). When considering biofilms and planktonic cells separately, no significant improvement between the use of two or more than two housekeeping genes was obtained (Fig. 4). The two most stably expressed control genes in C. albicans biofilm and planktonic cells separately were the ACT1 and RPP2B genes, and the RPP2B and PMA1 genes, respectively.

cDNA was amplified using a real time PCR MGB (Minor Grooving Binding) Taqman probe assay. The absolute C_t values from all the qPCR assays were used to calculate the expression ratios of the ALS1 and ALS3 genes in C. albicans biofilm cells compared to their planktonic counterparts. Table 3 shows the mean expression ratios of the ALS1 and ALS3 genes in C. albicans biofilm cells calculated with the four different normalization strategies (i.e., using the five or three most stably expressed HK genes [5HK or 3HK], ACT1 as a control gene and cDNA input standardization, respectively). These ratios are based on the expression of those two genes in all the biofilm samples compared to all the planktonic samples. Statistical analysis shows a significant upregulation of the ALS1 gene in C. albicans biofilm cells (p < 0.05). When using different normalization methods, we found that the ALS1 gene expression was three-fold (5HK genes and cDNA input), four-fold (3HK genes) or five-fold (ACT1) induced in C. albicans biofilms compared to planktonic cells. In contrast, there appeared to be a significant downregulation of the ALS3 gene in C. albicans biofilms (p < 0.05). We found a fourteen-fold (5HK genes), twelve-fold (3HK genes), seven-fold (ACT1) or ten-fold (cDNA input) downregulation of the ALS3 gene.

We also compared the ALS1 and ALS3 gene expression ratios obtained with the four different normalization strategies. No statistically significant difference in ALS1 and ALS3 gene expression ratios was observed when using the geometric mean of the relative expression levels of three or five housekeeping genes nor with standardization of the cDNA input. However, normalization using ACT1 as a single reference transcript resulted in higher expression ratios, both for ALS1 and ALS3 compared to the other strategies (p < 0.01).

C. albicans strain SC5314, which was kindly provided by Dr. A. Brown (Aberdeen University, UK), was used throughout this study. Cells were stored frozen at -80°C using the Microbank system (Prolab Diagnostics). For every experiment a fresh culture was prepared by taking two beads from a Microbank vial and transferring them into 10 ml Sabouraud Dextrose Broth (SDB; BD). This culture was incubated for 24 hours at 37°C. For culturing planktonic cells and biofilms, 50 μl of this suspension was added to 10 ml SDA and incubated for 16 hours at 37°C in a water bath with shaking.

C. albicans biofilms were grown on silicone disks (Q7-4735; Dow Corning) in the CDC biofilm reactor (Biosurface Technologies) 26 according to the protocol described by Honraet et al. 27, with some modifications. Cells were harvested by centrifugation, washed three times with and resuspended in 0.9% NaCl w/v. The resulting suspension (containing approximately 10⁸ CFU/ml) was added to 500 ml Yeast Nitrogen Base (YNB; BD) supplemented with 50 mM glucose. This inoculated medium was transferred to the CDC reactor and incubated at 37°C on a magnetic stir plate (RET digi-visc, IKA Labortechnic) set at 80 rpm for 24 hours, to allow cells to adhere to the silicone disks. After 24 hours, 0.2X YNB supplemented with 10 mM glucose was pumped through the reactor for 24 hours at a flow rate of 400 ml/hour. After 24 hours of biofilm growth, the disks were taken out of the reactor and each disk was transferred to 0.9% NaCl w/v. Prior to RNA extraction, disks were subjected three times to 30 s of sonication (Branson 3510, 42 kHz, 100 W, Branson Ultrasonics Corp.) and 30 s of vortex mixing. This treatment is necessary to remove the biofilm cells from the silicone 28.

After 16 hours of growth, the suspension was centrifuged and the pellets were washed three times with and resuspended in 0.9% NaCl w/v. Subsequently, 40 μl of this suspension was added to 10 ml of YNB broth supplemented with 50 mM glucose and incubated for 24 hours at 37°C in a water bath with shaking. After 24 hours the cells were centrifuged and resuspended in 10 ml diluted YNB (0.2X YNB supplemented with 10 mM glucose). These tubes were incubated for 24 hours at 37°C in a water bath with shaking. After 24 hours incubation, the cells were harvested by centrifugation and resuspended in 0.9% NaCl w/v. Prior to RNA extraction, this 1 ml suspension was subjected to vortex mixing and sonication, as described above.

The cell suspensions were centrifuged and the supernatant was discarded. Total RNA was extracted with the Ambion Pure Yeast kit (Ambion) according to the manufacturer's instructions. Isolated RNA was DNase treated with the DNase treatment kit (Ambion) to ensure the absence of genomic DNA contamination. Further purification of total RNA was carried out with the Microcon columns according to the manufacturer's instructions (Millipore). Total RNA was quantified prior to cDNA synthesis using the RNA Quant-it kit (Invitrogen) according to the manufacturer's instructions.

First strand cDNA synthesis was performed using the Superscript II kit (Invitrogen). Five μg of total RNA and 1 μl oligodT12-18 primer (0.5 μg/μl) were added to each tube to obtain a total volume of 12.5 μl. Priming was carried out at 70°C for 20 min. Subsequently, 2 μl 10× RT buffer (200 mM Tris-HCl pH 8.4 and 500 mM KCl), 2 μl 25 mM MgCl₂, 1 μl 10 mM dNTP, 2 μl 0.1 M DTT, 1 μl Superscript II RT (50 units/μl) and 0.5 μl RNaseOut (Recombinant Ribonuclease Inhibitor 40 units/μl) were added to each reaction tube. The RT reaction was carried out at 27°C for 10 min, followed by 50 min at 42°C and finally 5 min at 90°C (Amplitron Thermolyne). Subsequently, 1 μl of Escherichia coli RNaseH (2 units/μl) was added to each reaction tube and the tubes were incubated for 20 min at 37°C. cDNA was quantified using the OligoGreen Single Stranded Quantification kit (Invitrogen) according to the manufacturer's instructions.

Full-length gene sequences were obtained from the C. albicans database http://www-sequence.stanford.edu/group/candida/search.html29. Primers and MGB Taqman probes were designed using the Primer Express software (Applied Biosystems) according to the manufacturer's instructions. The primer and MGB Taqman probe sequences of all the genes used in this study are listed in Table 4. These sequences were compared to the C. albicans database using BLAST 30 in order to determine their specificity. Primers and MGB Taqman probes which could have resulted in non-specific signals were excluded. Eight housekeeping genes were selected based on different criteria. First, genes which are commonly used as a control for real time PCR experiments in C. albicans were included. Secondly, genes which appeared to be differentially expressed in biofilm and planktonic cells in the micro-array study of Garcia-Sanchez 9 were excluded. Furthermore, to avoid co-regulation of gene expression, the housekeeping genes tested were selected to belong to different functional categories. The control genes tested in this study were ACT1, PMA1, RPP2B, TDH1 (GAPDH), LSC2, CPA1, IMH3 and RIP.

Real time PCR was performed in 96 well plates with the ABI 7000 apparatus (Applied Biosystems) using the MGB Taqman probe assay. The concentrations of the primer and MGB Taqman probes used in this study are listed in Table 4. Unless otherwise specified, 5 μl cDNA was added to each reaction. Alternatively, the amount of cDNA was standardized and 40 ng cDNA was added to each reaction. Each reaction contained 12.5 μl Taqman Universal PCR Mastermix in a total volume of 25 μl. The real time PCR reactions were performed at 95°C for 10 min, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. For each sample a four-point standard curve was made with serial two-fold dilutions (1, 1/2, 1/4 and 1/8). Primer efficiency was determined for every sample/gene combination of all the gene expression assays using the formula E = 10^-1/slope, with "slope" being the slope of the four-point standard curve. Control samples were included on multiple plates to ensure that different plates could be compared. In addition, the samples were subjected to electrophoresis on 2.5 % agarose gels (Invitrogen) in 1× TBE buffer (1 M Trizma Base, 0.9 M boric acid and 10 mM EDTA, pH 8.4; Sigma) for 90 minutes at 100 V. Subsequently, gels were stained with ethidium bromide to confirm the presence of the expected PCR products and the absence of unwanted non-specific products.

Gene stability analysis of different housekeeping genes was performed using the geNorm VBA applet 23. For every control gene, the expression stability (M) was calculated as the standard deviation of the logarithmically transformed expression ratios. This M value is the average pairwise variation of one particular gene compared to all the other control genes. The gene with the lowest M value is considered as the most stable gene and the gene with the highest M value is excluded. A new M value is calculated and this procedure is repeated until only two genes are left. These two genes have the lowest M value and are therefore most stably expressed in all the samples.

Evaluation of the number of control genes required for accurate and reliable normalization was performed by calculation of the pairwise variation (V) between ranked normalization factors (NF). NFs, based on the expression values of the most stable housekeeping genes (lowest M value), were calculated using the geometric mean. For each combination of two sequential normalization factors (NF_n and NF_n+1) the pairwise variation (V_n/n+1) was calculated (n being the number of housekeeping genes tested). A large variation between two sequential NF's means that the added gene has a significant effect on the calculated normalization factor and should therefore be included in the calculations used for normalizing the data. The cut-off value, below which the inclusion of an additional control gene was considered not to result in a significant improvement of the normalization, was set at 0.15.

ALS1 and ALS3 gene expression in C. albicans biofilms was evaluated using four different normalization strategies. Gene expression data were normalized using (i) the geometric mean of the five most stably expressed housekeeping genes (5HK genes), (ii) the geometric mean of the three most stably expressed housekeeping genes (3HK genes), (iii) ACT1 gene as a single reference transcript (ACT1), and (iv) standardization of the cDNA input (cDNA input). The normalized expression ratios of the ALS1 and ALS3 genes using 5HK genes or 3HK genes were calculated according to the procedure described by Vandesompele et al. 23. Over- or underexpression of these two genes relative to the ACT1 gene was calculated as described by Pfaffl 31. ALS1 and ALS3 gene expression data obtained by normalization of the input cDNA were calculated using the delta delta C_t method 32.

Statistical analysis was performed using the SPSS 11.0 software (SPSS). One-way ANOVA was used to compare the four different normalization strategies.

A one-sample two-tailed t-test was used to determine whether differences in ALS1 and ALS3 gene expression between C. albicans biofilms and planktonic cells were statistically significant.