Normal human osteoblasts and MCF7 mono and coculture

As a model for investigating changes in gene expression in response to epithelial-osteoblast interactions in bone metastases of breast carcinomas, cells that represented malignant epithelial cell compartments (MCF7) and cells that represented skeletal compartments (Normal Human (NH) Osteoblasts) were examined in an in vitro mixed co-culture setting. These two types of cells were co-cultivated for 48 h in a low-serum medium [0.2% fetal bovine serum (FBS)] to allow reciprocal signal exchange with minimal background from undefined molecular signals that are inherent in fetal bovine serum. We examined the effects of co-cultivation on each cell pairing in two independent biological replicates. The gene expression profiles of the co-cultures were compared to the expression profiles of the corresponding cells that were kept in monoculture using HEEBO microarrays. After mono or co-culturing, total RNA was extracted and amplified using a modified Eberwine procedure. The amplified RNA was labeled with the fluorescent dye Cy5 and pooled with Cy3 labeled reference RNA, and then the pooled RNA was hybridized onto HEEBO microarrays. After hybridization and washing, arrays were scanned on a fluorescent microscope scanner. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.