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| Status |
Public on Oct 16, 2014 |
| Title |
AJ-mice_liver_control_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
AJ-mice, liver, control, replicate 3
|
| Organism |
Mus musculus |
| Characteristics |
strain: A/J
tissue: liver
gender: Male
age: 20 weeks
|
| Treatment protocol |
Male A/J, C3H/HeJ and WSB/EiJ were housed in sterilized cages in a temperature-controlled room (24°C) with a 12-h light/dark cycle, and given ad libitum access to water and NIH-41 irradiated pelleted diet. At 8 weeks of age, mice from each strain were allocated randomly to control and experimental groups. Mice in the methyl-donor-deficient experimental groups were maintained on a diet low in methionine (0.17% w/w) and lacking choline and folic acid (diet no. 519541, choline- and folate-deficient, iron-supplemented, and l-amino acid-defined diet; Dyets, Bethlehem, PA, USA) for 12 weeks. Mice in the methyl-adequate control groups received the same diet supplemented with 0.4% methionine, 0.3% choline bitartrate, and 2 mg/kg folic acid. Experimental and control mice were euthanized by exsanguination following deep isoflurane anesthesia 12 weeks after diet initiation. The liver was snap-frozen immediately in liquid nitrogen and stored at −80 °C for subsequent analyses.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from liver tissue using RNeasy Mini kits (Qiagen, Valencia, CA) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-2000C spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the Quick-Amp Labeling kit (Agilent Technologies) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Mouse GE Oligo Microarrays (G4858A-028005) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression in the livers of A/J mice fed a control diet
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. The raw data were normalized using ArrayTrack software (USFDA National Center for Toxicological Research, Jefferson, AR).
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| |
|
| Submission date |
Oct 15, 2014 |
| Last update date |
Oct 16, 2014 |
| Contact name |
Volodymyr Tryndyak |
| E-mail(s) |
volodymyr.tryndyak@fda.hhs.gov
|
| Phone |
870-543-7545
|
| Organization name |
US-FDA National Center for Toxicological Research
|
| Department |
Division of Biochemical Toxicology
|
| Street address |
3900 NCTR Rd
|
| City |
Jefferson |
| ZIP/Postal code |
72079 |
| Country |
USA |
| |
|
| Platform ID |
GPL13912 |
| Series (1) |
| GSE62362 |
Interstrain differences in the severity of liver injury induced by a choline- and folate-deficient diet in mice are associated with dysregulation of genes involved in lipid metabolism. |
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