Custom cDNA Microarray Hybridization Protocol

For the self versus self hybridizations, custom cDNA microarray experiments proceed as follows. Altogether, three microarray hybridizations were performed using custom printed cDNA microarray slides from the same print batch. Labelling, hybridization and washing were done as described previously by Monni et al. [40 (link)] and Järvinen et al. [20 (link)]. Briefly, total RNA was extracted from cell lines BT-474, HBL-100, and MCF-7 and labelled with Cy3-dUTP and Cy5-dUTP (Amersham Biosciences, Piscataway, NJ). The custom printed cDNA microarrays comprised of 11,520 clones from Incyte Genomics IRAL cDNA library and 1,136 clones from Research Genetics library. Microarrays were printed on poly-l-lysine coated slides using an Omnigrid arrayer (GeneMachines) as described previously [20 (link)]. Microarrays were scanned with an Agilent laser confocal scanner (Agilent Technologies, Palo Alto, CA) and gridded using the DEARRAY software developed by Chen et al. [29 (link)]. For the four breast cancer cell lines, custom cDNA microarray experiments were provided in a separate contribution by Järvinen et al. [20 (link)] and detailed protocols are described in that work. The relevant genes in our study were verified using RT-PCR in a parallel study by Hyman et al. [35 (link)].