title: Rainbow trout custom 8.8k microarray
status: Public on Dec 29 2007
contact.city: Nantes
contact.first: Daniel
contact.last: BARON
contact.email: daniel.baron@univ-nantes.fr
contact.state: Loire-Atlantique (44)
contact.address: 30 Bd Jean Monnet
contact.department: UMR1064 - Centre de Recherche en Transplantation & Immunologie
contact.country: France
contact.institute: Institut national de la santé et de la recherche médicale-INSERM
organism: Oncorhynchus mykiss
manufacture_protocol: DNA Microarray construction : Gene expression analyses were carried out using home-made Nylon microarrays using a previously described technology [10441335]. These microarrays were built using as templates cDNA clones provided by the INRA-AGENAE program [16887034]. All these clones were PCR-amplified at the INRA Resources Centre for Animal Genomics (CRB GADIE, Jouy en Josas, France) using primers designed on the plasmid sequences flanking the cDNA inserts (M13RP1 5’- GTGGAATTGTGAGCGGATAAC and M13RP2 5’-GCAAGGCGATTAAGTTGGG). PCR amplifications (35 rounds) were carried out in 100μl of 1X buffer containing 25 mM MgCl2, 250 μM dNTP, 100μM each primer, and 2.5 Units of Taq polymerase (Promega, Madison, WI). For each cDNA clone, two amplification products were pooled, desiccated and resuspended in 50 μl of distilled water. They were then spotted as previously described [10445855] onto Hybond-N+ 2x7 cm2 membranes (Amersham Pharmacia Biotech, Cleveland, OH, USA) stuck to glass slides using a 8-pin print head (Pin-and-Ring™ technology) on the GMS 417™ (Affymetrix, MWG-Biotech, Ebersberg, Germany). Spotted DNA was then denatured and UV– cross-linked onto nylon filters. All membranes used in this study were made at the same time and under the same condition.