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Sample GSM269542 Query DataSets for GSM269542
Status Public on Mar 01, 2008
Title 1month_60/0 (060711_04)
Sample type RNA
 
Source name liver of 4-5 weeks (young) and 12-14 month (adult) old mice
Organism Mus musculus
Characteristics 1month_60/0
Treatment protocol Sham treatment; 30, 60 or 90 minutes of partial hepatic ischemia
Extracted molecule total RNA
Extraction protocol The median and left lobe of the liver were dissected from young (4-5 weeks) and old (12-14month) mice that underwent either ischemia or Sham control operations and immediately submerged in RNAlater [Ambion, Inc., Austin, TX].
Quality control steps: Total RNA was purified using RNeasy columns [Qiagen, Valencia, CA] according to the manufacturer’s directions and quality was assessed using an Agilent 2100 Bioanalyzer [Agilent Technologies, Inc., Palo Alto, CA].
Label biotin
Label protocol 10 µg of total murine liver RNA from the 8 groups (n=3 of young Sham controls, old Sham control, young 30 min. of ischemia, old 30 min. of ischemia, 60 min. of ischemia, old 60 min. of ischemia, young 90 min. of ischemia, old 90 min. of ischemia) was amplified with the Genechip Expression 3’ Amplification One-Cycle Target Labeling and Control Reagents Kit (Affymetrix) to prepare biotin-labeled fragmented cRNA. Specifically, the One-Cycle cDNA Synthesis Kit (Affymetrix) was used to make double-stranded cDNA from total RNA. cRNA was labeled with biotin-UTP and biotin-CTP by an in vitro transcription reaction using the IVT Labeling Kit (Affymetrix).
 
Hybridization protocol Full Protocol Description: Create a hybridization cocktail for a single probe array that contains 0.067 µg/µL fragmented cRNA, 50 pM Control Oligonucleotide B2 (Affymetrix), 20X Eukaryotic Hybridization Controls (1.5 pM bioB, 5 pM bioC, 25 pM bioD, 100 pM cre) (Affymetrix), 10% DMSO, and 1X Hybridization Buffer. Heat hybridization cocktail to 99°C for 5 minutes, to 45°C for 5 minutes, and spin at maximum speed in a microcentrifuge for 5 minutes. Fill probe array with 200 µL of 1X Hybridization Buffer. Incubate at 45°C for 10 minutes in the GeneChip Hybridization Oven 640 (Affymetrix) rotating at 60 rpm. Remove 1X Hybridization Buffer and fill probe array with 200 µL of the hybridization cocktail. Incubate at 45°C for 16 hrs in the Hybridization Oven rotating at 60 rpm.
Fluidics: Wash and Stain probe arrays using the Fluidics Station 450 (Affymetrix) utilizing the fluidics protocol FS450-0001. Arrays were stained with R-Phycoerythrin-conjugated streptavidin [Molecular Probes, Eugene, OR] and hybridization signals were amplified using and anti-streptavidin antibody (goat) biotinylated [Vector Laboratories, Burlingame, CA], as described in the Affymetrix GeneChip® Expression Analysis Manual.
Scan protocol Images were scanned using a GeneChip Scanner 3000 [Affymetrix]
Description Sham Control and 30, 60 and 90 minutes of ischemia.
Data processing Affymetrix murine MOE 430 2.0 gene chips were used.GeneChip CEL files were subjected to RMA normalization using the GeneSpring GX 7.3.
Standard Affymetrix internal control genes were used to check the quality of the assay quality by the signals of the 3' probe set to the 5' probe set of the internal control genes, GAPDH and B-actin, with acceptable 3' to 5' ratios between1-3. Prokaryotic Spike controls were used to determine whether the hybridization of target RNA to the array occurred properly.
The data were analyzed with GeneSpring version 7.3 (Agilent Technologies, Palo Alto, CA) using RMA normalization with a custom CDF file(Mm430_Mm_REFSEQ_8]), then normalized to the mean of the sham samples of the same age
 
Submission date Feb 27, 2008
Last update date Feb 27, 2008
Contact name Bhuvaneswari Sakthivel
E-mail(s) bhuvana.sakthivel@cchmc.org
Phone 513-636-0146
Fax 513-636-2056
Organization name CCHMC
Department Biomedical Informatics
Street address 3333 Burnet Ave
City Cincinnati
State/province OH
ZIP/Postal code 45229
Country USA
 
Platform ID GPL5759
Series (2)
GSE10652 Divergent genome expression profiles during hepatic ischemia in young and adult mice
GSE10657 Hepatic response to ischemia and reperfusion: young and adult mice

Data table header descriptions
ID_REF
VALUE data was RMA normalized and then normalized to the mean of the sham samples of the same age

Data table
ID_REF VALUE
NM_001001144_at 0.8160705
NM_001001152_at 0.83003867
NM_001001160_at 1.2942592
NM_001001176_at 0.8863403
NM_001001177_at 1.1232394
NM_001001179_at 1.2862287
NM_001001180_at 0.9293855
NM_001001181_at 0.87247276
NM_001001182_at 1.1103809
NM_001001183_at 0.6623804
NM_001001184_at 0.8040379
NM_001001295_at 0.9420006
NM_001001297_at 0.93793756
NM_001001309_at 0.63227904
NM_001001326_at 1.0271434
NM_001001333_at 1.1164715
NM_001001335_at 0.9994324
NM_001001444_at 1.1728638
NM_001001446_at 0.88533866
NM_001001447_at 1.1470215

Total number of rows: 26180

Table truncated, full table size 598 Kbytes.




Supplementary file Size Download File type/resource
GSM269542.CEL.gz 3.5 Mb (ftp)(http) CEL
Processed data included within Sample table

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