Tissue: Testis parenchyma Age: 90 days Strain: Sprague-Dawley Exposure=650 mg methoxyacetic acid/kg body weight i.p. Time Post treatment: 24 hr Method of termination: CO2 asphyxiation Microarray ID: 251186831792 Hyb Date: 2005-06-29
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from a portion of the right testis (about 30 mg) using the Qiagen RNeasy mini kit (Qiagen, Mississauga, ON) following the manufacturerÆs directions, and quantified using Quant-iT RiboGreen RNA assay kit (Invitrogen, Burlington, ON). Total RNA quality was assessed using the RNA 6000 Nano LabChips with the 2100 Bioanalyzer system (Agilent Technologies Inc., Mississauga, ON, Canada). Only samples with the ratios of 18s/28s ribosomal RNA intensity ratios of between 1.65 and 2.0 were used for microarray analyses.
Label
Cy5
Label protocol
Individual total RNA samples of testis from each animal or universal rat RNA were labelled with Cyanine 5-CTP or Cyanine 3-CTP, respectively (Perkin Elmer Life Sciences, Woodbridge, ON, Canada) using Agilent linear Amplification kits (Agilent Tech. Inc.) following the manufacturerÆs instruction. Briefly, double-stranded cDNA was synthesized using MMLV-RT with T7 promoter primer, starting with 5 ╡g total RNA. Cyanine-labeled cRNA targets were in vitro transcribed using T7 RNA polymerase. The synthesized cRNA was precipitated by LiCl, yielding 25 ~35 ╡g labelled cRNA-target. Three micrograms of labelled cRNA were fragmented at 60║C for 30 min with fragmentation solution.
Channel 2
Source name
universal rat reference total RNA (Catalog #740200; Stratagene, La Jolla, CA, USA)
Total RNA derived from 14 rat cell lines Microarray ID: 251186831792 Hyb Date: 2005-06-29
Biomaterial provider
Stratagene, La Jolla, CA, USA
Extracted molecule
total RNA
Extraction protocol
Unknown: Material was purchased as total RNA (universal rat reference total RNA (Catalog #740200; Stratagene, La Jolla, CA, USA))
Label
Cy3
Label protocol
Individual total RNA samples of testis from each animal or universal rat RNA were labelled with Cyanine 5-CTP or Cyanine 3-CTP, respectively (Perkin Elmer Life Sciences, Woodbridge, ON, Canada) using Agilent linear Amplification kits (Agilent Tech. Inc.) following the manufacturerÆs instruction. Briefly, double-stranded cDNA was synthesized using MMLV-RT with T7 promoter primer, starting with 5 ╡g total RNA. Cyanine-labeled cRNA targets were in vitro transcribed using T7 RNA polymerase. The synthesized cRNA was precipitated by LiCl, yielding 25 ~35 ╡g labelled cRNA-target. Three micrograms of labelled cRNA were fragmented at 60║C for 30 min with fragmentation solution.
Hybridization protocol
Cy5- sample cRNA and Cy3-labelled common reference cRNA (rat universal reference total RNA (BD Biosciences Clontech, Palo Alto, CA, USA) were hybridized to Agilent rat oligo microarrays (containing ~20,000 unique 60 mer oligonucleotides; Agilent Tech. Inc.) at 60║C overnight with Agilent hybridization solution and washed according to the Agilent's instructions.
Scan protocol
Arrays were scanned on a ScanArray Express (Perkin Elmer Life Sciences, Woodbridge, ON, Canada), and data were acquired with ImaGene 5.5 (BioDiscovery, Inc. El Segundo, CA).
Description
MAA_MAA_24_251186831792.txt
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: a robust composite method addressing single and multiple slide systematic variation. 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 using the least square estimates.
The acute effects of methoxyacetic acid exposure on testis gene expression
Data table header descriptions
ID_REF
VALUE
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, ImageneFlag is the Imagene flag value, BackgroundFlag where 1 indicates if the raw median signal intensity is below the trimmed mean (5%) plus 3 trimmed standard deviations of the negative control spots, ProcessedCy3 is the normalized median signal intensity adjusted for the day of hybridization, ProcessedCy5 is the normalized median signal intensity adjusted for the day of hybridization
Flag
flag information; 1 = absent (not more than 3 S.D. above mean background); 0 = present
