Analysis of globle gene expression changes observed in fibroblasts isolated via explant outgrowth and 45-minute differential binding

Fibroblasts are a ubiquitous cell type that are found in many tissue types and are associated with a variety of diseases including most fibrotic disorders. We isolated fibroblast cells using explant outgrowth methodologies and compare the genomic signature of these cells to cells isolated using 45-minute differential binding. 14 total pulmonary fibroblast samples were used in this study. These fibroblasts were isolated from 4 lungs with end-stage idiopathic pulmonary fibrosis and 3 normal donors lungs (controls) designated brain dead, non-diseased but not suitable for transplantation. 8 IPF samples included 4 isolated via explant outgrowth and 4 isolated via 45 minute differential binding . Three normal samples included 3 isolated via explant outgrowth and 3 isolated via 45 minute differential binding. RNA extraction was carried out using Qiagen RNeasy Kit. Experimental/control samples were amplified amino-allylated RNA labeled with Cy5 and Stratagene Reference RNA was amplified and amino-allylated and labeled with Cy3. One round of amplification was carried out using Ambion MessageAmp II kit with amino-allylated UTP according to the protocol of the Duke University Institute for Genome Sciences and Policy. Amplification and amino-allylation of the Stratagene Reference RNA and Hybridization of Reference with all samples were carried out by the Duke Institute for Genomic Sciences and Policy.