status: Rapid age: 48 gender: male tissue: cultured human fibroblasts
Treatment protocol
No additional treatments were applied to the cultured cell lines
Growth protocol
Primary cultures of LFs were isolated from the distal parenchyma of patients from unaffected donor controls (lung cancer patients) and patients with IPF as previously described [Hogaboam CM, Carpenter KJ, Evanoff H, Kunkel SL (2005), Pechkovsky DV, Prele CM, Wong J, Hogaboam CM, McAnulty RJ, et al. (2012)]. All fibroblast cell lines were completely de-identified from the patients they were grown from prior to characterization in this study. The IPF lung tissue was obtained and the diagnosis of Usual Interstitial Pneumonia (UIP) / IPF achieved as previously outlined [Pechkovsky DV, Prele CM, Wong J, Hogaboam CM, McAnulty RJ, et al. (2012)]. Normal human lung tissue was obtained from macroscopically tumor-free lung resections of patients with lung cancer. The human lung tissue collection was approved by the ethics committees of all institutions involved. In this study we characterized fibroblasts cell lines originated from 14 donors across 3 phenotypes: non-IPF control (n = 4), stable IPF (n = 6), and rapidly progressing IPF (n = 4) subjects. Cells from these lines were plated and passaged (up to the 11th passage).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated and homogenized in QIAzol reagent. Purified RNA was then amplified and labeled using NuGen Ovation kits
Label
biotin
Label protocol
Purified total RNA was amplified and labeled using NuGen Ovation kits (NuGEN Technologies, Inc., San Carlos, CA) and RNA from samples was hybridized to Affymetrix HG-U133 plus 2.0 arrays.
Hybridization protocol
Array washing, staining and scanning was performed according to standard Affymetrix protocols .
Scan protocol
Array washing, staining and scanning was performed according to standard Affymetrix protocols .
Data processing
Probe level data was curated by first mapping individual probe sequences to their most current genome sequences. Probes which were non-uniquely mapped to specific genes or contained outdated mappings were discarded, and the remaining probes were summarized into probesets and normalized using Robust Multi-array Average (RMA). Probes expressed at low levels across all samples were subsequently discarded.
