title: Candida Albicans Eurogentec 13440
status: Public on Oct 01 2006
contact.city: Madrid
contact.first: Concha
contact.last: Gil
contact.state: Madrid
contact.address: Pza. Ramón y Cajal s/n
contact.department: Departamento Microbiología II
contact.country: Spain
contact.institute: Universidad Complutense de Madrid
organism: Candida albicans
manufacture_protocol: Construction of C. albicans MicroArray
  
  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.
  
  Spotting design
  
  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.
  
  MicroArray content
  
  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
  Quality control
  
  The C. albicans MicroArrays are certified to generate a high quality data.
  All printed slides are previously selected for coating uniformity and low intrinsic background.
  The MicroArrays are visually inspected for spotting quality (doughnuts), uniformity, integrity and for perfect alignment of spots and grids.
  From each batch of printed MicroArray, three are randomly chosen for further quality control. We use the Terminal Transferase reaction to assess the amount of DNA covalently attached to the slides. Finally, we perform a real hybridization using labeled cDNA obtained from heat-choc cultured cells.