LREG, CRB_GADIE, INRA Jouy en Josas, France, F_78350
Manufacture protocol
The SLA/PrV microarray, composed of cDNA and sub-cloned exons, is a dedicated array containing porcine and PrV probes. This microarray was constructed in the CRB-GADIE (Centre de Ressources Biologiques-Génomique des Animaux Domestiques et d'Intérêt Economique, LREG, INRA, Jouy-en Josas, France). The list of genes present in the human SLA orthologous region (6p21-p23) was drawn up using the human sequence draft (May 2004) on UCSC web site (http://genome.ucsc.edu/). Corresponding porcine cDNA clones were selected when they were available in the AGENAE library or in 2 American libraries MARC1PIG and MARC2PIG constructed in US by the United States Departement of Agriculture (USDA). When no porcine cDNA clone was found, exons were identified by comparison between ESTs and genomic DNA using ICCARE comparison mapping tool (http://genopole.toulouse.inra.fr/Iccare/). Primers were designed with Primer 3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi).PrV amplicons (80) targeting 70 genes an pig exons were amplified by PCR and subcloned (PGEMT easy plasmid, Promega and XXX). cDNA and sub-cloned exons were amplified by PCR (Taq PCR Master Mix Kit, Qiagen) with universal primers located in the vector (35 cycles:30 sec at 94°C, 30 sec at 60°C and 2 min at 70°C). After purification (Multiscreen-PCR plates, Millipore), PCR products were checked on 1% agarose gel, quantified (Fluoroscan), evaporated and pellets were resuspended in 13µl SSC3X. Forty six control spots were added on the slide (Lucidea Universal scorecard, Amersham Biosciences). Two identical arrays were spotted on slides (25x75 mm, UltraGAPS Coated Slides, Corning) with a 16 needles spotter (Microarraywriter Pro,Virtek). After spotting, slides were first treated with steam to homogenise spots hydratation. Then spotted DNA was denatured (5 sec at 100°C) and UV fixed (300 mJ). Slides were stored in dry atmosphere before use. All the SLA and immune clones spotted on the SLA/PrV slides were sequenced. Sequence homology was checked by multi-alignement (BLAST) against human and pig EST database (NCBI and VEGA). The SLA/PrV microarray probes were reannotated taking into account the most significant BLAST results and his final geneID file was used in the analysis.
Support
Glass (25x75 mm, UltraGAPS Coated Slides, Corning)
Coating
Aminosilane
Description
Two identical networks per slide were spotted. A network contains 16 blocks with 12 rows and 12 columns for each block.
Less...
More...