Tissue: part of lung Gender: Male Age: 8-10 week C57BL/6 x CBA F1 hybrid mice Date of exposure: 2005-02-16 Exposure: restrainers only Time post exposure: 3 h micro array ID:30025 Hybridization Block 2
Extracted molecule
total RNA
Extraction protocol
RNA was isolated using the TRIzol reagent (Invitrogen Canada Inc., Burlington, ON, Canada), and further purified using RNeasy Mini Kits (Qiagen Inc., Mississauga, ON, Canada). RNA was quantified using UV spectrophotometer, and quality was confirmed using the Agilent 2100 Bioanalyzer and RNA 6000 NanoLab Chip Kit (Agilent Technologies Canada Inc., Mississauga, ON, Canada).
Label
Cy5
Label protocol
Individual 2.5 µg aliquots of RNA from each sample were amplified and labelled using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent, (5184-3523)).
Channel 2
Source name
universal mouse reference total RNA (Catalog #740100; Stratagene, La Jolla, CA, USA)
Total RNA derived from 11 mouse cell lines micro array ID:30025 Hybridization Block 2
Biomaterial provider
Stratagene, La Jolla, CA, USA
Extracted molecule
total RNA
Extraction protocol
Unknown: Material was purchased as total RNA (universal mouse reference total RNA (Stratagene, CA, USA))
Label
Cy3
Label protocol
Individual 2.5 µg aliquots of RNA from each sample were amplified and labelled using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent, (5184-3523)).
Hybridization protocol
Agilent Mouse G4121A Microarrays (containing approximately 22,000 probes) were hybridized with 5 µg Cy5-labeled lung RNA and 5 µg Cy3-labeled Universal Mouse Reference RNA (Stratagene, CA, USA), used as a common reference on all arrays. Arrays were incubated overnight at 60 ºC in Agilent hybridization solution and washed according to manufacturer’s instructions.
Scan protocol
Arrays were scanned using a ScanArray Express (Perkin-Elmer Life Sciences, Woodbridge, ON, Canada), and data were acquired with ImaGene 5.5 (BioDiscovery, CA, USA).
Description
Ratio is the normalized log2 ratio (sample/reference), RawCy3 is the raw median signal intensity for the reference, RawCy5 is the raw median signal intensity for the sample.
Data processing
A blocked factorial design, using the date of hybridization and the date of exposure, was used to analyse lung microarray data. The data were normalized using a LOWESS curve using the R MAANOVA library. Ratio intensity plots and heat maps for the raw and normalized data were also constructed using R. Differentially expressed genes were determined using the MAANOVA library in R. The main effects in the model included treatment, duration of exposure and break period, as well all two-way and the three-way interaction. This model was applied to the log2 of the relative intensities. The Fs statistic, a shrinkage estimator for the gene-specific variance components, was used to test main effects, interactions and pair-wise comparisons. The p-values for all statistical tests were estimated by the permutation method using residual shuffling, followed by adjustment for multiple comparisons by using the false discovery rate (FDR) approach.
