title: CLI/Metagenomics_Gut_739_v5
status: Public on Jan 01 2010
contact.city: Brisbane
contact.first: Seungha
contact.last: Kang
contact.state: QLD
contact.address: 306 Carmody road
contact.country: Australia
contact.institute: CSIRO
organism: Bacteria
  Archaea
manufacture_protocol: A custom phylogenetic microarray consisting of eubacterial and archaebacterial probes was designed based on 16S rRNA sequences from gastrointestinal bacteria. Initially, a search for bacterial sequences previously identified as gastrointestinal microbes was performed on the Entrez Nucleotide database. Individual probes were then designed using the GoArray oligonucleotide program. In addition, probes obtained by way of a literature search of published papers were included, as well as several reference probes which served as internal standards and for normalization of the data. The reference probes targeted different sequences within the rrs human mitochondrial gene (positive controls), while probes which targeted sequences outside this region acted as negative controls for determining the level of background signal. Furthermore, the probes were also checked for specificity using the ‘Probe Match’ function of ARB software. Thus, the oligonucleotide probes designed for the microarray represented diverse taxonomic groups, with specificities ranging from species to phylum levels but the majority were species specific. The final oligonucleotide probes, as well as several negative and quality control probes were synthesized onto a 4 X 2K custom microarray by Combimatrix (Mukilteo WA, USA) with three replicates of each probe randomly distributed across the array to reduce biases caused by spatial variations.