Human dermal endothelial cells exposed to 10% human control sera Cells were plated in T75 flasks and grown to subconfluence and serum was removed at designated time point and cells were rinsed three times with PBS prior to extraction of RNA for microarray studies. The cDNA from HDEC exposed to SCL70 SSc sera and that from Human Universal RNA (Stratagene, La Jolla, California) as a common probe across all the arrays (reference-experimental design) was covalently coupled with separate Cy5 and Cy3 monoreactive fluors. RNA was extracted from the cell cultures using TRIzol (Invitrogen, Carlsbad, California). The RNA was DNase I-treated (Invitrogen) and purified on an RNeasy column (Qiagen, Valencia, California) with purity being assessed by UV absorbance (260/280 ratio >1.8) and by agarose gel analysis. We utilized the hapten-antibody method (Micromax TSA kit, Perkin-Elmer, Boston, Massachusetts) for labeling of the probes with Cy3 and Cy5 which required a minimum of ~5 mg of input total RNA. After drying, the array images were acquired on a confocal dual-laser scanner (Lumonics ScanArray Lite, Perkin-Elmer) equipped with Scanarray and Quantarray software (Perkin-Elmer). Background corrected intensity expression levels were normalized per channel using Cluster as follows: one cycle of log transformation, five cycles of median centering of genes, and five cycles of normalizing genes to generate a log-transformed, median polished, and normal (magnitudes close to 1.0) dataset. Arrays (representing sequential serum incubation times) were not adjusted. Hierarchical correlation (centered) complete linkage clustering was performed of genes using Cluster and illustrated in heat maps by TreeView. Pathway Analysis: a Gene Microarray Pathway Profiler (GenMAPP) http://www.genmapp.org/) was utilized to visualize gene expression data on maps representing biological pathways and groupings of genes pertinent to apoptosis, angiogenesis, and autoantigens. Fold ratios were calculated by dividing normalized gene expression data for HDEC exposed to either control sera or SSc sera (at 1, 6, 12, and 24 hours) by normalized gene expression data for HDEC exposed to control sera for 5 minutes. A difference of three-fold was arbitrarily chosen to enable GenMAPP to assign colors representing upregulation and downregulation in pathways of biological significance and narrow the number of genes analyzed to < 500. The intention was to utilize Cluster, TreeView, and GenMAPP to qualitatively illustrate the patterns of gene expression induced in HDEC by sequential serum stimulation. Triplicate sets of data were averaged and experimental data was normalized by dividing with respective spot intensity for reference RNA Keywords = human dermal endothelial cell, scleroderma
