RNA from Candida albicans CAI4-URA grown on bare plastic for 90min
Extracted molecule
total RNA
Label
Cy3
Description
Preparation and labeling of the cDNA and hybrid¬ization with the microarrays were performed following standard protocols (http://microarrays.org). Briefly, 10μg of total RNA was reverse transcribed with Superscript II reverse transcriptase (Invitrogen, Carlsbad, Calif.) in the presence of 5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphate (aa-dUTP), using both oligo dT and random primers (Stratagene, La Jolla, Calif.). The cDNAs from the control and experimental conditions were coupled with Cy3 or Cy5 monoreactive dyes (Amersham Biosciences, Piscataway, N.J.), mixed at 1:1 ratio, and concentrated in a Microcon-30 spin column (Millipore, Billerica, Mass.). The concentrated probes were hybridized with the microarray slide in Hybridization Buffer #3 (Ambion, Austin, Tx.) at 50°C for 16 h. The arrays were visualized with 428TM array scanner (Affymetrix, Santa Clara, Calif.). At least three hybridizations were performed for each time point, host cell type, and strain of C. albicans.
Data processing
The scanned images of both channels were quantified using ImaGene 5.0 (Biodiscovery, El Segundo, Calif.). Spots were quantified as the median value of all pixel intensities in the spot region. The local background values were calculated from the area surrounding each feature and subtracted from respective spot signal values. The data from each array were normalized by locally weighted linear regression (lowess) analysis using the MIDAS program (http://www.tigr.org/software/).
