title: Schistosoma mansoni microarray v1
status: Public on Aug 24 2006
contact.city: São Paulo
contact.first: Katia
contact.middle: Cristina Pereira
contact.last: Oliveira
contact.state: O
contact.address: Av Prof lineu prestes 748
contact.department: Centro de Parasitologia e Micologia
contact.country: Brazil
contact.institute: Instituto Adolfo Lutz
organism: Schistosoma mansoni
manufacture_protocol: We have constructed a 9,216-element microarray (4,608 unique elements in duplicate) using EST clones from a large scale sequencing of the S. mansoni transcriptome [Verjovski-Almeida et al., Nature Genet. 35:148-157, 2003]. We used the assembly of S. mansoni ESTs produced by this large-scale project (available at http://bioinfo.iq.usp.br/schisto/) for data mining and selection of relevant transcripts to be spotted. After sorting out the transcripts of interest (see below), we selected for each transcript the 3'-most clone from its cluster. These clones were amplified using PCR in combination with universal primers designed from the cloning site vector sequences flanking the inserts.  PCR products were purified using MultiScreen PCR 96 filter plate (Millipore), following manufacturer instructions. Successful amplification and purification of products were verified by agarose gel electrophoresis stained with ethidium bromide. Purified products eluted in 50 mM phosphate buffer pH 8.0 were spotted on Type 7-star glass slides (GE Healthcare), using a Generation III Microarray System (GE Healthcare).  A non-redundant set of 4,088 probes representing different SmAEs [Verjovski-Almeida et al., Nature Genet. 35:148-157, 2003] plus positive and negative controls was selected for array construction, using both manual and automatic methods.  We selected and mannually curated 1,104 probes, each one representing a gene with a biological function of interest as determined by comparing the translated S. mansoni EST contig sequences (using the BLASTx tool and a cutoff E-value of e-10) to the GenBank protein database and the Gene Ontology (GO) annotation bank. Among these, there are probes representing 290 genes involved in protein and amino acid metabolism, 50 in carbohydrate metabolism, 5 in RNAi, 20 in iron metabolism, 10 in purine and pyrimidine metabolism, 40 in lipid metabolism, 50 in DNA repair, 70 in transporters, 45 in cellular adhesion, 15 in apoptosis, 70 in cell cycle, 30 in protein processing and 150 in signaling processes and receptors. We also selected 696 probes automatically from genes coding for S. mansoni orthologs (among them 241 annotated as hypothetical or unknown). Selection criteria were: 1) a BLASTx search in GenBank (cutoff E-value e-10) shows that they match to proteins of other organisms; 2) no GO terms associated; 3) the sequence comes from a SmAE contig (not a singlet); 4) best hit must not be a bacterial protein. In addition, there are 1,155 probes for Schistosoma gene transcripts with the highest protein-coding scores as determined by ESTscan but no-match by BLASTx search against GenBank, which probably code for proteins of yet unknown function, and 1,133 probes for Schistosoma genes without protein-coding potential and no-match by BLASTx search against GenBank. The last line of each of 12 subarrays contained a set of Lucidea Universal scorecard controls (GE Healthcare), which include 2 negative controls per set, totalizing 48 negative control spots per slide that were used as a cutoff value for filtering out low intensity gene signals. Each probe in the array is spotted in duplicate, at the left and right halves of the slide, totalizing 9,216 spots per slide.
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  =======================ANNOTATION UPDATE - May 2007 ===============================
  We have updated the automatic annotation of our platform.  The controls (424) and manually curated genes annotations remain unaltered (1143 genes). The automatic selected genes are now categorized on: automatically selected with functions (656), automatically selected with no function (hypothetical) (867). The unkown genes are categorized on: Unknown coding (645) and unknown non coding (873), based on ESTscan results.