title: UofG_Mallard_Bovine_Immune-endocrine_v2.0
status: Public on May 19 2007
contact.city: Guelph
contact.first: Maria
contact.middle: Isabel
contact.last: Nino-Soto
contact.email: mnino@uoguelph.ca
contact.state: Ontario
contact.address: College Ave. West
contact.department: Pathobiology
contact.country: Canada
contact.institute: University of Guelph
contact.phone: 519-8244120
organism: Bos taurus
description: The cDNA microarray is defined as 4 replicate blocks were each block consists of 20 columns and 21 rows, with features (PCR products, positive and negative controls) printed in duplicate. The microarray is printed twice on the slide surface.
manufacture_protocol: This platform is based on a previously developed cDNA microarray (Tao et al, 2004, Vet Immunol Immunopathol. 101(1-2):1-17). The present version consists of 183 RT-PCR derived DNA fragments, of which 20 represent new sequences with respect to the previous version (GPL4473). The microarray includes 5 housekeeping genes and bovine genomic DNA as positive controls as well as Rhodococcus equi 17kD virulence protein gene, plasmid pACYC177 DNA and printing buffer as negative controls. Total RNA was extracted from several sources including bovine blood mononuclear cells, spleen, ovaries and placenta. Total RNA was reverse transcribed with Superscript III (Invitrogen Canada Inc., Ontario, Canada) and sequences were amplified from cDNA with gene-specific primers. PCR products were cloned using the TOPO TA Cloning® kit for Sequencing (Invitrogen Canada Inc., Ontario, Canada). PCR products amplified from cell lysates using M13 primers were purified using the 384 Well DNA Binding UNIFILTER® (Whatman Inc., New Jersey, USA) according to the manufacturer’s instructions. Purified PCR products were dehydrated by lyophilization and resuspended in H2O/DMSO [1:1] for printing. Printing was performed on Corning® UltraGAPS™ Coated Slides (Corning Inc., New York, USA) using a VersArray ChipWriter Pro robot (Bio-Rad Laboratories (Canada) Ltd., Ontario, Canada). Slides were incubated in a dessiccator for 48 hours before UV cross-linking with 300 mJ. Slides were heat-sealed back into their original storage pouch to protect them from light and humidity changes. Quality control was performed with The PARAGON™ DNA Microarray QC Stain Kit (Invitrogen Canada Inc., Ontario, Canada).