Twenty-five micrograms of total RNA was labeled in a total volume of 40 µl including 5 ng LuxA control RNA (Eurogentec), 1x first-strand buffer (Invitrogen), 0.2 pmol C. albicans-specific primer (Eurogentec), 1.5 mM (each) dATP, dTTP, and dGTP, 25 µM dCTP, 10 mM dithiothreitol, 1 µl RNasin, and 37.5 µM Cy5-dCTP (Amersham). The mixture was incubated at 65°C for 5 min and then at 42°C for 5 min. One microliter of RNasin (Promega) and 1 µl Superscript II reverse transcriptase (Invitrogen) were added and incubated at 42°C for 1 h. Following the addition of another 1 µl of Superscript II, the incubation was continued for 1 h at 42°C. The reaction was stopped by the addition of 5 mM EDTA, pH 8.0, and 0.5 M NaOH and incubation at 65°C for 20 min and was neutralized by adding 0.5 M acetic acid. The labeled cDNAs were purified using a QIAquick PCR purification kit (QIAGEN) and concentrated by drying under vacuum to a volume of approximately 2 µl.
As for Channel 1, but using 37.5 µM Cy3-dCTP (Amersham)
Hybridization protocol
Before hybridization, one of the labeled cDNA samples was resuspended in 45 µl of hybridization buffer (1x Dig Ease buffer [Roche] containing 0.5 mg ml–1 baker's tRNA and 0.5 mg ml–1 salmon sperm DNA) and then added to the second labeled cDNA. The mixture was heat denatured at 95°C for 2 min, quickly cooled on ice, applied to the DNA microarray, and covered with a 24-mm by 60-mm coverslip. Slides were placed in a hybridization chamber (Corning, Palo Alto, CA) with 9 µl of 3x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) in each of the wells and incubated at 37°C for 16 h by immersion in a water bath. Following hybridization, the slides were immersed in 1x SSC at 50°C until the coverslip fell off, washed twice for 15 min each in 1x SSC-0.1% sodium dodecyl sulfate (SDS) at 50°C and once in 0.1x SSC-0.1% SDS for 20 min at 50°C, and rinsed three times in 1x SSC at 50°C and, finally, once in 0.2x SSC at room temperature. The slides were dried by centrifugation at 900 rpm for 5 min. Seven independent comparisons (incorporating three dye swaps) were performed for yeast growth conditions, and five independent comparisons (with two dye swaps) were performed for growth under hypha-inducing conditions.
Scan protocol
The microarrays were scanned using an Axon 4000B scanner at a 10-µm resolution
Description
Transcriptional comparison of WT and ACE2 knockout strains grown in the yeast form
Data processing
Low-quality spots were automatically flagged by Genepix Pro 5.0. Lowess normalization was carried out in GeneSpring.
Transcriptional comparison of C. albicans WT and ACE2 knockout strains
Data table header descriptions
ID_REF
VALUE
Log2 (normalised Cy5/Cy3 ratio)
CH1_MEDIAN_RAW
The median intensity value calculated from total amount of pixels of the spot in channel 1
CH1_BKD_MEDIAN_RAW
The median intensity value calculated from total amount of local background pixels of the spot in channel 1
CH2_MEDIAN_RAW
The median intensity value calculated from total amount of pixels of the spot in channel 2
CH2_BKD_MEDIAN_RAW
The median intensity value calculated from total amount of local background pixels of the spot in channel 2
CH1_INTENSITY_MINUS_BKD
Intensity minus background for channel 1
CH2_INTENSITY_MINUS_BKD
Intensity minus background for channel 2
FLAGS
Gives information about the data depending on the value of the flag. Flag 0 means unflagged. Flag -50 means spot not found when GenePix align blocks fails to align a spot indicator
RATIO_NORM
Ratio of normalised intensities. The ratio is calculated by dividing channel 1 by channel 2 (Cy5/Cy3).
