For genome-wide transcriptional analysis of C. albicans challenged by the presence of lactobacilli, cocultures of both microorganisms were established in 6-well plates with ThinCert (Greiner Bio-One, Monroe, NC, USA) inserts that kept both cell populations separated by a PET membrane with a pore-size of 0.45 μm. Fungal cells were inoculated in the lower compartment and pregrown for 6 hrs at 37°C in 5% CO2 atmosphere without shaking. Lactobacilli were then inoculated in the ThinCert compartment and the cocultures were incubated up to 24 hrs total incubation time (i.e., 18 hrs in coculture). Parallel C. albicans cultures without addition of lactobacilli served as controls. At various time points, the pH of the medium was measured and the cell density of both bacterial and fungal cells was determined by spectrophotometry at 600 nm. C. albicans cells were harvested and total RNA was isolated using a FastPrep FP120A instrument in conjunction with the FastRNA Pro Red kit (MP Biomedicals, Solon, OH, USA). Isolated total RNAs underwent further purification with the Qiagen RNeasy Mini kit (Qiagen, Valencia, CA, USA). Integrity and concentration of the total RNAs were determined by agarose gel electrophoresis and spectrophotometry, respectively. For preparation of Affymetrix GeneChip (Affymetrix, Santa Clara, CA, USA) hybridization targets, 50 ng of the highly-purified total RNAs were used for cDNA generation, Ribo-SPIA amplification, and biotin-labeling using the NuGEN Ovation Biotin system (NuGEN Technologies Inc., San Carlos, CA, USA). The resulting biotin-labeled single-stranded cDNAs were used in C. albicans GeneChip hybridizations according to the manufacturers' directions. The custom-made C. albicans GeneChips were described previously [12]. Following hybridization in a GeneChip Hybridization Oven 640 and a GeneChip Fluidics Station 400 (Affymetrix), the microarrays were scanned using a GeneArray Scanner (Hewlett-Packard). The resulting image files were processed for absolute and comparative expression analysis using MICROARRAY SUITE 5.0 (MAS 5.0; Affymetrix). Global scaling to a target intensity of 500 was used to correct for variations between arrays. Experimental data were stored in MICRODB 3.0 and analyzed in DATA MINING TOOL 3.0 (Affymetrix) as well as MICROSOFT ACCESS. Default parameters were used for the statistical algorithms implemented in MAS 5.0 to calculate probe set signals, signal log ratios (SLR) as well as detection and change P values. For determination of differential gene expression between Lactobacillus-Candida coculture experiments and Candida-only controls, we also employed the SNOMAD software for standardization and normalization of microarray data (http://pevsnerlab.kennedykrieger.org/snomadinput.html [13]). Here, signals (intensities) of experiment-control sets are log-transformed and the log ratios are normalized by calculation of a local mean across the element signal intensities (LOESS fit). Furthermore, a local variance correction is applied and a Zscore is calculated for each probe set as a measure for differential expression. Transcripts with Z scores ≥3 were considered significantly increased and transcripts with Z scores ≤−3 were considered significantly decreased in comparative analyses. In order to group genes with similar regulation, cluster analyses were performed using the programs Cluster and TreeView [14].
μm. Fungal cells were inoculated in the lower compartment and pregrown for 6