title: Eurogentec-Scerevisiae-Array
status: Public on Dec 01 2007
contact.city: Montpellier cedex1
contact.first: Jean-Luc
contact.last: Legras
contact.email: jean-luc.legras@inrae.fr
contact.address: 2, place Viala
contact.department: UMR1083, Science pour l'Oenologie
contact.country: France
contact.institute: INRA
organism: Saccharomyces cerevisiae
manufacture_protocol: * 5803 yeast ORFs spotted in duplicate on glass slide
  * 68 control genes
  * Also available:
  - Set of primer pairs for PCR
  - Set of aminated PCR products
  - Mix of specific oligos for RT reaction
  - Optimized hybridization
  Construction of S. cerevisiae MicroArray
  Comparative genome analysis of 6213 predicted Saccharomyces cerevisiae open reading frame (ORF) products with 13 yeast genome sequence related species proposed the elimination of 612 predicted ORFs that are not real genes (A. Malpertuy et al, FEBS Letters, Volume 487, 22 December 2000). The study identified the presence of 50 new genes.
  Specific pair of primers was designed by Pasteur Institute, Paris to amplify all 5803 ORFs non-spurious coding sequences. 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 agarose gels.
  Spotting design
  The yeast 5803 ORFs MicroArray is composed of 32 subgrids, which follow a pattern of 8 metarows and 4 metacolumns. Each subgrid contains 20 columns and 21 rows with a dot spacing of 200 µm. Probes are spotted as closed duplicates. In order to provide a friendly environment, the first subgrid row on each array metarow contains all the normalization controls, which include the controls with serial dilution of the Renilla LuxA cDNA PCR product. In addition to this control, four serial dilutions of the sonicated yeast S288c genomic DNA are also located in the corner subgrids of the MicroArray.
  MicroArray content
  To determine the background values, the stringency of the hybridization and washing conditions we add the following probes to the MicroArray:
  1- Three E. coli genes showing no homology with yeast ORFs (aceF, kdtA and tufA),
  2- Ten yeast intergenic regions
  3- Three orthologous genes from Schizosaccharomyces pombe Leu1, His1 and Ura4 and printing buffer are spotted on the MicroArray (negative controls).
  Marker genes are also present onto the MicroArray in order to follow the expression of genes of interest (Schistosoma mansoni 28 kDa glutathione S-transferase, Aequorea victoria green-fluorescent protein GFP, TAP fusion tag, human c-myc tag, LexA DNA binding domain , Pho4 DNA binding domain, b-galactosidase LacZ 5'- and 3'- encoding regions) or the genetic background of a KanMX-modified strain.
  Finally, two sets of oligonucleotides cover the large genes YKL182w (6.153 kbp, 11 oligonucleotides), and YLR310c (4.567 kbp, 9 oligonucleotides) to allow the user to monitor the efficiency of the reverse transcription reaction.
  Quality control
  * The S. cerevisiae MicroArrays are certified to generate 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 MicroArrays, 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 from heat-shock cultured cells.
  Packaging