cell type: FE1 cells from lung time of growth: Cells were grown for 72 hrs growth conditions: 24hrs prior to exposure, the cells were washed and incubated with serum free media passage: Cells of passage 14 at 70% confluence date of exposure: 2007-22-02 exposure: 6 hours exposure to 4 mg/ml of coal tar diluted in DMSO incubation: 8 hours id: Microarray ID Cy5 48 cell line: FE1
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). Material was purchased as total RNA (universal mouse reference total RNA (Stratagene, CA, USA)
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)
sample type: Total RNA derived from 11 mouse cell lines source: Stratagene, La Jolla, CA, USA
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). 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).
Hybridization protocol
Agilent Mouse G4121B Microarrays (containing approximately 22,000 probes) were hybridized with 5 µg Cy5-labelled FE1 cells (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
T8D2
Data processing
The median signal intensity of each microarray sample was used. Technical replicates were not averaged. Data for each probe was flagged (flag=1) if the median signal intensity for that probe was less than the mean of the negative controls ((-)3xSLv1) plus three standard deviations. RawCy5 and RawCy3 are the median signal intensities for the Cy5 and Cy3 channels on each microarray. NormCy5 and NormCy3 are the loess normalized signal intensities for the Cy5 and Cy3 channels within each array; this is a within-array normalization performed separately for each array. Flag indicates whether the median signal intensity falls in the background, i.e below threshold calculated from the negative controls (mean plus three standard deviations of the negative controls.) Ratio is calculated as log2(NormCy5/NormCy3).
