90 minutes of partial hepatic ischemia and 1 hour of reperfusion
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/reperfusion or Sham control operation 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 4 groups (n=3 of young Sham controls, old Sham control, young 90 min. of ischemia/ 1 hour of reperfusion, old 90 min. of ischemia/ 1 hour of reperfusion) 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/L 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), 0.1 mg/mL Herring Sperm DNA (Promega), 0.5 mg/mL Acetylated BSA (Invitrogen), 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 EukGE-WS2v5_450. Arrays were stained with phycoerythrin-conjugated streptavidin [Molecular Probes, Eugene, OR] and hybridization signals were amplified using antibody amplification with goat IgG [Sigma-Aldrich] and anti-streptavidin biotinylated antibody [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 90 minutes of ischemia followed by 1 hour reperfusion.
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
