title: CRG Human Breast Cancer Array v4.0-0.8K
status: Public on Oct 05 2007
contact.city: barcelona
contact.first: BELEN
contact.last: MINANA
contact.email: belen.minana@crg.es
contact.state: barcelona
contact.address: dr aiguader 88
contact.department: GENE REGULATION, STEM CELLS AND CANCER
contact.country: Spain
contact.institute: Center for genomic regulation
contact.phone: 0034933160276
organism: Homo sapiens
manufacture_protocol: 826 cDNA clones corresponding to genes possibly involved in breast cancer were selected to constitute our Breast Cancer Array (BCA) cDNA platform. In this platform there are different interlinked biological pathways represented: cell cycle, DNA repair, DNA damage, apoptosis, DNA remodeling, PR related pathways, endometrial and ovarian cancer related genes, and prognosis genes. Clones were selected from the German Human Genome Resource Center (http://www.rzpd.de/) from different cDNA of endometrial, ovary or breast cDNA libraries, and a clone information database was constructed using several web resources such as SOURCE (http://source.stanford.edu/cgi-bin/source/ sourceSearch, Diehn et al. 2003). The selection criterion was to choose the most 3’ end clone, which contained a polyadenylation signal with a maximum length of 2 kb. We visualized these features and confirmed the position of the clone within its cluster using GeneNest graphical database (http://genenest.molgen.mpg.de). All cDNA clones were confirmed by sequencing from the 5’-end, and their position within the gene was determined by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/).
  DNA plasmid preparations were generated using Plasmid miniprep 96 kit (Millipore; cat.no.LSKP09604). Inserts were PCR amplified using a homemade thermostable Thermus aquaticus (Taq) DNA polymerase clone (Desai and Pfaffle, 1995), with vector specific primers. PCR products were purified with PCR 96 Cleanup kit (Millpore; cat.no.LSKC09604). PCR products were normalized to 100 ng/uL and allocated on 384-well plates. PCR products were printed in 2 x Corning Pronto! ® spotting solution on CORNING UltraGAPS® II amino-modified glass slides, using a Robotic arrayer ChipWriter (Bio-Rad) and a pin-head of 16 SMP3 printing needles (Telechem®). cDNA spots were immobilized by UV crosslinking at 2500 mJ/s with an UV oven and stored under vacuum at desiccators until they were used for hybridization.
  Each gene product was printed in quadruplicates on each microarray, two of them side-by-side on the same row, and the other two in different subgrids, by inverting the spotting plates, on the top or bottom halve of the array.