Dataset features


Application: Gene expression microarray analysis
Number of samples: 8
Release date: Jul 8 2008
Last update date: Mar 25 2013
Access: Public
Diseases: Infertility, Polycystic Ovary Syndrome, Yellow Fever
Chemicals: Androgens, Thiazolidinediones
Computational protocol: GenePix Pro, GeneSpring GX
Dataset link Pioglitazone treatment of Lethal Yellow mice

Experimental Protocol

Experimental design: Female heterozygous Lethal Yellow (C57BL/6J Ay/a) mice were randomized to vehicle treatment group (0.1 ml 100% DMSO, n=4) or pioglitazone treatment group (0.1 mg pioglitazone in 0.1 mg 100% DMSO). The drug/vehicle was administered by gavage daily from 120 days to 180 days of age. The mice were euthanized on day 60 of treatment (180 days of age) and the ovaries were removed and placed in RNAlater (Ambion). Quality control steps: The cRNA that was synthesized from each mouse ovary was used for hybridization to a single CodeLink (Amersham/GE) whole mouse microarray. Only one sample was hybridized with each slide and only one dye (Alexa 647) was used so no dye swaps were necessary. Bacterial control spikes were used as per manufacturer's instructions. Samples used, extract preparation and labelling: The origin of each biological sample: The samples were ovarian tissue obtained from heterozygous Lethal Yellow (C57BL/6J Ay/a) mice that had been treated by gavage with vehicle (DMSO) or pioglitazone (0.1 mg/day) for 60 days. Manipulations of biological samples and protocols used: The ovaries were dissected free from the animals, cleaned of adhering fat, placed in RNAlater, and frozen at -80C until RNA was extracted. Experimental factor: drug treatment Technical protocols: The ovaries were homogenized in TRI reagent (MRC) using a Polytron (Brinkmann). The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen). The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen). The purity and quantity were evaluated by an Agilent Bioanalyzer using the RNA 6000 Nanoassay LabChip. Labelled cRNA was prepared using the manufacturer's Instruction Protocol (, CodeLink Gene Expression System: Manual Labelled cRNA Target Preparation). The source of reagents was the CodeLink Expression Assay Reagent Kit, Manual Prep, except where specified otherwise. 2.0 micrograms of total ovarian RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C. (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.) The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C. The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C. The double-stranded cDNA was purified on QIAquick columns (Qiagen) and the eluent was dried down in a SpeedVac concentrator. The double-stranded cDNA was resuspended in a mixture containing T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA. The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on RNeasy columns (Qiagen). The concentration of cRNA was determined by spectrophotometry. Hybridization procedures and parameters: 10 micrograms of cRNA was mixed with fragmentation buffer and heated to 94C for 20 min. The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides, and hybridized for 18 hours at 37C. The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature. The Alexa fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer) The slides were then washed 4 times for 5 min each in 1x TNT and twice in 0.05% Tween 20 for 5 sec each. The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.








Kathleen Eyster

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