Profiling of human bone marrow stromal cell lines HS-5 and HS-27a

The microarrays were constructed using a set of more than 17,000 sequence-verified clones from Research Genetics (Huntsville, AL). Of the 17,761 features (spots) on the microarray, 186 are control nonexpressed or nonhuman sequences or housekeeping genes. UniGene cluster IDs could be assigned by GenBank to 16,592 of the features as of June 1, 2001 (UniGene Build 133), indicating that they are representatives of nonredundant unique genes. Many have been functionally characterized, and chromosomal location and tissue expression patterns are known for others. Total RNA was isolated from semiconfluent cultures and reverse-transcribed into cDNA in a nucleotide mix containing amino-allyl deoxyuracil triphosphate (dUTP). The cDNA from stromal cells and Universal RNA (Stratagene, La Jolla, CA) was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively. The Universal RNA consists of a mixture of RNA from 10 different human cell lines with a broad expression coverage of over 80% of the sequences on the array, allowing comparison of expression patterns of multiple different samples of different origin. The Cy5- and Cy3-labeled cDNAs were combined for hybridization to the microarray. Fluorescent array images were collected for both Cy3 and Cy5, and image-intensity data were extracted and analyzed to obtain expression ratios to Universal RNA for each stromal cell line. From these the expression in the 2 lines could be compared. Keywords: other