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| Status |
Public on Mar 26, 2021 |
| Title |
Liver_E2F2_HFD_rep4 |
| Sample type |
RNA |
| |
|
| Source name |
Liver, E2F2 knockout mice, diethylnitrosamine treatment, high fat diet, replicate 4
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Liver
genotype: E2F2 Knockout
treatment: Dietylnitrosamine
diet: High fat diet
|
| Treatment protocol |
WT (129/Sv x C57BL/6), E2f1-/- and E2f2-/- mice were administered a single injection of DEN (25 mg/kg of mice, Sigma-Aldrich, USA) intraperitoneally on postnatal day 14. After weaning at 1 month old, mice were fed a HFD (60 % fat calories mouse diet, F3282, Bio-Serv, USA) for 32 weeks and sacrificed at 9 months old. Mice were fasted for 4 hours and were euthanized with sodium pentobarbital (Abbott Laboratorios, Spain) injected intraperitoneally (60 mg/kg of mice in PS). Liver was collected and washed in cold PBS. Then, it was frozen in liquid nitrogen and stored at -80 °C until analysis were performed.
|
| Growth protocol |
5-6 mice were placed in each cage and were housed at 22 °C with an artificial 12 hours light (8:00 to 20:00)/12 hours dark cycle. Food and water were provided ad libitum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from liver homogenates using Trizol Reagent (Invitrogen, USA) and according to the manufacturer instructions.
|
| Label |
Cy3
|
| Label protocol |
RNA samples were labeled and hybridized following standard Agilent Protocol One-Color Microarray-Based Gene Expression analysis (Low Input Quick Amp Labeling) v 6.5. Briefly, 50 ng of total RNA were retrotranscribed and labeled using Low imput Quick Amp Labeling kit one color (Agilent Technologies) following manufacturer instructions. First, total RNA was retrotranscribed with AffinityScript Reverse Transcriptase (Agilent Technologies), using Oligo dT primers coupled to T7 promoter. Double stranded cDNA synthesized by AffinityScript RT was in vitro transcribed by T7 RNA pol in the presence of Cy3-CTP to generate amplified and labeled cRNA. Labeled samples are purified with silica-based RNeasy spin columns (Qiagen, Hilden, Germany). After labeling and purification, cRNA was quantified with NanoDrop 1000 spectrophotometer in order to determine the yield and specific activity of each reaction.
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| |
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| Hybridization protocol |
Samples were analysed using Agilent SurePrint G3 Mouse GE 8x60K microarrays (Design ID 028005) (Agilent Technologies), with 55,681 mouse features represented. 600 ng of labeled cRNA samples were hybridized at 65 ºC for 17 h in a SureHyb hybridization chamber (Agilent Technologies).
|
| Scan protocol |
The arrays were scanned immediately after washing on a G2565CA DNA microarray scanner, with ozone-barrier slide covers (Agilent Technologies) using one-color AgilentG3_GX_1color scanner protocol for HD microarray formats (scanner settings Dye channel: Green, Scan Region: Agilent HD (61x21.6 mm), Scan resolution 3 um, Tiff file dynamic range 20 bit, Green PMT gain 100 %)
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| Description |
Hepatic gene expression after E2F2 knockout mice were exposed to diethylnitrosamine and high fat diet
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software v 10.7.3.1. (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 028005_D_F_20140728) to obtain background subtracted and spatially detrended Processed Signal intensities. Raw data from Feature Extraction software was subsequently processed on GeneSpring GX 12.5 (Agilent Technologies). GeneSprings transforms all FE Flags to a single Detection Flag and calculates the geometric mean of replicated probes.
Data were normalized by quantile normalization and centered by mean.
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| |
|
| Submission date |
May 20, 2020 |
| Last update date |
Mar 26, 2021 |
| Contact name |
Patricia Aspichueta |
| E-mail(s) |
patricia.aspichueta@ehu.eus
|
| Organization name |
University of the Basque Country UPV/EHU
|
| Department |
Physiology
|
| Street address |
Barrio Sarriena s/n
|
| City |
Leioa |
| ZIP/Postal code |
48940 |
| Country |
Spain |
| |
|
| Platform ID |
GPL10787 |
| Series (1) |
| GSE117420 |
Gene expression profile in E2F1 and E2F2 deficient mice treated with the procarcinogen agent diethylnitrosamine and fed a high fat diet |
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