The mice were housed with free access to food and water under conditions of 12 h of darkness and 12 h of light. After an overnight fast, the animals were killed and the lung tissues were rapidly dissected and flash frozen in liquid nitrogen. Tissue samples were obtained from two adult male mice for each strain in the RCS panel. The snap frozen tissue samples were homogenized in TRIzol reagent using a polytron, and RNA was prepared according to the manufacturer's instructions.
Label
biotin
Label protocol
cRNA probes were prepared from 10 µg of total RNA dissolved in 10 µL of DEPC-treated water and 1 µL (100 pmol) of T7- (T)24 primer (Genosys, GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG- (T)24) was added. The primer-RNA mixture was denatured for 10 minutes at 70C, and then chilled on ice. First strand cDNA synthesis was performed using 2 µL of Superscript II reverse transcriptase (Invitrogen Canada Inc.) in a 20 µL reaction volume containing 10 µM DTT, 500µM each dNTP and 1x First Strand Buffer (all Invitrogen Canada Inc.) for 60 minutes at 42C. Second strand synthesis was performed by adding 40U DNA Polymerase I, 10U E. Coli DNA ligase, 2U RNAse H in a final reaction volume of 150 µL containing 1x Second Strand Buffer (all Invitrogen Canada Inc.). The reaction was incubated at 16C for two hours and stopped by adding 10 µL of 0.5 M EDTA (Sigma). Following second strand synthesis, the probe cDNA was purified by phenol chloroform extraction using Phase-Lock tubes (5’-3’) and redissolved in 20 µL of DEPC-treated water. Biotinylated probe was prepared from the entire cDNA reaction using the ENZO Bioarray High Yield RNA Transcript Labeling Kit (ENZO diagnostics). The probe synthesis reaction was performed at 37C for 5 hours with occasional agitation. The biotinylated probe was purified using an RNeasy spin column (Qiagen), eluted in 80 µL of DEPC-treated water, quantified by spectrophotometry and probe quality was assessed using Agilent BioAnalyser RNA LabChips. The average probe length was reduced by incubating the purified probe in 1x Fragmentation Buffer for 35 minutes at 95C.
Hybridization protocol
The hybridization mixture was prepared by mixing 15 µg of biotinylated probe with Control Oligonucleotide B2 (final concentration 50 pM) (Affymetrix), Herring Sperm DNA (final concentration 0.1 mg/ml) (Research Genetics), Acetylated BSA (final concentration 0.5 mg/ml) (Invitrogen Canada Inc.) in a final volume of 300 µL of 1x MES Hybridization Buffer (100 mM MES, 1M NaCl, 20mM EDTA, 0.01% Tween-20) (all reagents from Sigma). The hybridization mix was denatured for 10 minutes at 99ºC, incubated for 5 minutes at 45ºC and spun for 5 minutes in a benchtop microcentrifuge. The microarray expression studies were performed using an Affymetrix MG-U74Av2 GeneChips (Affymetrix) for each labeled RNA sample. The microarrays were warmed to room temperature and prehybridized in 1x hybridization buffer for 10-20 minutes at 45ºC. The prehybridization solution was removed and 150 µL of the hybridization mix was added to each array. The array and probe fragments were incubated at 45ºC overnight (16-20 hours). Following hybridization, non-specifically bound probe was removed by washing using the GeneChip Fluidics Station 400 (Affymetrix). In total, ten low stringency washes (6x SSPE, 0.01% Tween-20, 0.005% Antifoam) and four high stringency washes (100 mM MES, 0.1 M NaCl, 0.01% Tween-20, 50ºC) were performed (all reagents from Sigma). Detection of specifically bound probe was performed by incubating the arrays with SAPE (streptavidin phycoerthryin)(Molecular Probes) and biotinylated anti-streptavidin antibody (Vector Laboratories) and scanning the chips using a Gene Array Scanner (Agilent).
Scan protocol
The Chips were analyzed with an Affymetrix Gene Array Scanner (Agilent) controled by the Microarray Analysis Suite 5.0 (Affymetrix).
Description
Gene expression data from mouse whole lung tissue
Data processing
The R software package was used for all analyses. Custom CDFv12 was used on the raw expression data to deal with problem probesets. Expression values were normalized using the Robust Multi-array Analysis (RMA) for Affymetrix gene chips. To define the association between differentially expressed genes and genotypes, ANOVA was conducted on a per-gene basis using the linear model Expression ~ DSO + BG + DSO*BG, where background (BG) and donor strain of origin (DSO) were coded as binary phenotypes corresponding to A/J or B6. The cutoff for genome-wide significance was computed using the Benjamini–Hochberg correction.
