Spearman rank correlation analysis was first performed for a total of 5,560 common transcripts that passed the RNA-seq and microarray filtering criteria (detailed in Methods).
PANP (Presence-Absence Calls From Negative Strand Matching Probesets) 21 bioconductor R package was used for microarray filtering of background signals from unspecific hybridization (lower P value = 0.02 and upper P value = 0.01 for absent/marginal/present calls).
Four genes linked to asthma or lung inflammation (Arg1, Ccl24, Slc7a2, Mcpt1) that did not pass all the microarray filtering criteria were confirmed by qRT-PCR as being up-regulated in ESO versus ASO mice, which is consistent with an enhanced lung response in ETS-exposed mice.
Two copies of each rat GeneFilters cDNA microarray filter set (set A and set B; each comprised of gene filter releases gf300, gf301 and gf302) were screened in duplicate with labelled cDNA derived from independent preparations of RNA from adenovirus E1-transformed and untransformed BRK cells.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-2-microglobulin (B2M) were selected as normaliser genes from the initial microarray filter gene expression data.
Platform: Research Genetics (Invitrogen) - GF211 Microarray Filter Mendelian disorder: Marfan syndrome (MFS, OMIM #154700) Disease gene: fibrillin-1 precursor (FBN1, OMIM *134797) Phenotype: variable skeletal abnormalities, tall stature, disproportionately long limbs and digits, joint laxity, eye anomalies and progressive cardiovascular problems.
Platform: Research Genetics (Invitrogen) - GF211 Microarray Filter CF Mendelian disorder: Cystic fibrosis (CF, OMIM #219700) Disease gene: Cystic fibrosis transmembrane conductance regulator (CFTR, OMIM *602421, [43]) Phenotype: chronic obstructive lung disease, bronchiectasia, and exocrine pancreatic insufficiency Expression data: Wright et al. (2006) [13] (GEO accession number GDS2143).
A total of 18 hybridizations were done on six nylon cDNA microarray filters (GF211; ResGen).
After all hybridizations of the microarray filter had been performed, we stained the filter with a fluorescent dye that is specific for nucleic acids.
In addition, the 15,481 unique genes screened by the microarray filters used in this study represent 62% of the rat genome.
