Tissue: Whole lung Gender: Male Age: 127 days Strain: SP-C/TNF-α Date of exposure: 2004-10-27 Exposure: 50 mg/m3 EHC Time post exposure: 24 h Microarray ID: 251197830484 Hybridization Block 5
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 the RiboGreen RNA Quantitation Reagent and Kit (Molecular Probes, Eugene, OR, USA), 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).
Channel 2
Source name
universal mouse reference total RNA (Catalog #740100; Stratagene, La Jolla, CA, USA)
Total RNA derived from 11 mouse cell lines Microarray ID: 251197830484 Hybridization Block 5 Stratagene, La Jolla, CA, USA purchased from Stratagene
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 the RiboGreen RNA Quantitation Reagent and Kit (Molecular Probes, Eugene, OR, USA), and quality was confirmed using the Agilent 2100 Bioanalyzer and RNA 6000 NanoLab Chip Kit (Agilent Technologies Canada Inc., Mississauga, ON, Canada).
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).
Hybridization protocol
Agilent Mouse G4121A Microarrays (containing approximately 22,000 probes) were hybridized with 5 µg Cy5-labelled lung RNA and 5 µg Cy3-labelled 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
n/a
Data processing
The data were normalized by lowess curve (Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP. Normalization for cDNA microarray data:. Nucleic Acids Res. 2002; 30: e15.) using SAS/STAT software, Version 8.2 of the SAS System for Windows (1999 2001 SAS Institute Inc., Cary, NC, USA). Data used to estimate foldchanges were adjusted for variation due to date of hybridization and date of exposure using the least square estimates.
