title: PorkChip 2,600 cDNA array
status: Public on Oct 30 2004
contact.city: St. Paul
contact.first: Kendra
contact.middle: A.
contact.last: Hyland
contact.email: hyla0021@umn.edu
contact.state: MN
contact.address: 1971 Commonwealth Ave.
contact.department: Veterinary and Biomedical Sciences
contact.country: USA
contact.institute: University of Minneosta
contact.phone: 612-624-0622
organism: Sus scrofa
description: Spotted cDNA array on glass.
  This set includes approximately 2,600 cDNA clones, selected from a Peyer’s patch subtracted library and other sources.
  The PorkChip2 cDNA microarray contains 2,423 ESTs from the subtracted jejunal Peyer’s patch library (UMNMPM3), 10 ESTs from the normal jejunal Peyer’s patch library (UMNMPM1), and 26 ESTs from the immune-stimulated jejunal Peyer’s patch library (UMNMPM2), which were cloned using the GeneTrapper cDNA Positive Selection System and pCMVSport6 vector (Invitrogen Corporation, Carlsbad, CA) (2).  Each array also contains 86 ESTs identified by differential display RT-PCR (DD-RT-PCR) of PRRSV-infected macrophages (Library: PRRSV DD-RT-PCR)(5), 19 ESTs identified by DD-RT-PCR of influenza-infected porcine epithelial PK-15 cells (Library: UMNMPM4), 40 porcine genes, which were the result of directed cloning efforts (Library: Sus scrofa gene), and 7 porcine reproductive and respiratory syndrome virus strain VR-2332 sequences (Library: PRRSV) positive and negative controls (Library: Control).  Positive controls included 8 Ambion ArrayControl sequences. The plasmid inserts were amplified by two rounds of nested PCR using vector-specific primers.  The products from the second round PCR were cleaned by the Montage PCR(96) kit (Millipore, Billerica, MA) and were run on 0.8% agarose gels to quantify the amount of DNA by comparison to standards run on each gel.  The dried PCR products were resuspended in phosphate buffer (150 mM sodium phosphate and 1 mM EDTA (pH 8.5)) at a final concentration of 100-300 ng/microliter and transferred to 384 well plates. A BioRobotics MicroGrid II (BioRobotics Ltd., Haslingfield, Cambridge, UK) arrayed the DNA on slides coated with poly-L-lysine (Sigma, St. Louis, MO)(3).  Elements were arranged in 48 subarrays of 16 X 16, for a total of 12,288 spots per microarray, with each clone represented in by 4 spots.  The slides were baked for 15 min at 80 C, UV irradiated (65 mJ), and chemically blocked with 1-methyl-2-pyrrolidinone and succinic anhydride (4) and were stored at room temperature under vacuum until use.  The quality of each printing run was verified by staining one slide with SYBR Gold (Molecular Probes, Eugene, OR) and scanning at 488/530 nm (Excitation/ Emission) (1).
  Platform_references: 1. Battaglia C, Salani G, Consolandi C, Bernardi LR, and De Bellis G. Analysis of DNA microarrays by non-destructive fluorescent staining using SYBR green II. Biotechniques 29: 78-81, 2000.  2. Dvorak CMT, Hyland KA, Machado JG, Zhang Y, Fahrenkrug SC, and Murtaugh MP. Gene discovery and expression profiling in porcine Peyer's patch. Vet Immunol Immunopathol, submitted, 2004.  3. Eisen MB and Brown O. DNA arrays for analysis of gene expression. Methods Enzymol 303: 179-205, 1999.  4. Shalon D, Smith SJ, and Brown PO. A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization. Genome Res 6: 639-645, 1996.  5. Zhang X, Shin J, Molitor TW, Schook LB, and Rutherford MS. Molecular responses of macrophages to porcine reproductive and respiratory syndrome virus infection. Virology 262: 152-162, 1999.
  Keywords = small intestine, cDNA, pig