title: Eurogentec Candida albicans 6k microarray (condensed)
status: Public on Feb 22 2008
contact.city: Jena
contact.first: Bernhard
contact.last: Hube
contact.email: bernhard.hube@hki-jena.de
contact.address: Beutenbergstr. 11a
contact.country: Germany
contact.institute: Hans Knoell Institute
organism: Candida albicans
description: C. albicans is an opportunistic yeast that causes severe infections in immunocompromised individuals. Infections can be localized, such as vaginal infections and oral infections, which cause a considerable degree of discomfort. In some patient groups, whose defense system is severely compromised (premature infants, leukemics, burnt persons, and Aids patients), the yeast can turn into a deadly pathogen causing systemic infections up to 50 % of the patients infected die as a result. Although fluconazole has been an effective drug for a number of years, resistance is increasing. Alternatives such as amphotericin B have serious drawbacks, including such side effects as nephrotoxicity and severe discomfort. Few new antifungals are on the horizon, and more knowledge about the mechanism of pathogenicity of fungi as well as about their general biology is crucial if new drug targets are to be identified.
  MicroArray content (the physical array contains ~13k data features):
  * 98 % of C. albicans genes were spotted twice in a single glass slide
  * Tags (GFP, TAP, GST)
  * Marker genes Kan, Leu2, LacZ
  * Luciferase gene to spike mRNA samples
  * Negative controls (intergenic regions)
  * Cross hybridization S. cerevisiae genes having 70 %, 50 % and 30 % sequence identity with S. pombe genes cDNA quality controls
  * Dynamic range controls
  * Spatial hybridization controls
  
  The GPL6453 data table is a condensed representation of the physical microarray (~13k features).
manufacture_protocol: The C. albicans MicroArray has been developed in collaboration with the European Galar Fungail Consortium (www.pasteur.fr/recherche/unites/Galar_Fungail/).
  The Stanford Genome Technology Center generated the nucleotide sequence data for C. albicans with fundings from the NIDCR, NIH and the Burroughs Wellcome Fund. Information about coding sequences and proteins were obtained from the CandidaDB database: www.pasteur.fr/recherche/unites/Galar_Fungail/CandidaDB/, which has been developed by the Galar Fungail European Consortium.
  Specific primers were designed to amplify all 6039 putative ORFs. The primers were selected carefully to amplify only a specific region of each ORF.
  Universal sequences of 15 bases were incorporated on the 5' of each specific forward and reverse primer.
  The two-step PCR method has been employed to generate 5' amino-modified PCR for covalent attachment to the aldehyde-coated support. All the amplicons have an average length of 300 bp and they were controlled systematically by electrophoresis on 2 % agarose gels. Two independent probes per gene were selected to represent 34 genes. The MicroArray covers nearly 98 % of the annotated C. albicans genes and includes twelve mitochondrial genes, five intergenic regions, five S. cerevisiae genes and five genes for hybridization quality control, human glycerol-3-phosphate dehydrogenase and actin gene. We include in the MicroArray probes to evaluate the dynamic range of signal intensities, reverse transcription efficiency and spatial hybridization.
  The C. albicans MicroArrays are manufactured by printing PCR amplicons suspended in optimized spotting buffer for high coupling efficiency of DNA to the most consistent aldehyde coated glass slides. This spotting chemistry provides us the highest hybridization intensities, which is a consequence of a better binding of the probe to the slides and their accessibility to the cDNA targets. We use ChipWriter™ Pro (Virtek) which is a high precision dispensing robot to spot the PCR products onto aldehyde glass slides. The robot is designed to collect 100 nl of DNA solution and to deposit 0.6 nl per spot. For C. albicans MicroArray, we use simultaneously 32 pins to place the spots in area of 3.6 x 1.8 cm. In order to generate high quality spots, the printing procedure is performed under a tight controlled humidity and temperature environment. This allows having the complete control on the spot morphology, the spot diameter and uniformity. This configuration of the robot generates 32 blocks, each containing 20 x 20 dots with 200 microns spacing center to center with a spot diameter of 100 microns.