|
| Status |
Public on Apr 06, 2017 |
| Title |
HFD control, normoxia, replicate 5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
HFD control, normoxia, replicate 5
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
gender: male
age: adult
tissue: epididymal white adipose tissue
|
| Treatment protocol |
C57BL/6JOlaHsd wildtype male mice were acclimatized in the facility by feeding purified low fat diet (BIOCLAIMS, Hoevenaars et al., Genes and Nutrition 2012) for 3 weeks, followed by 12 weeks BIOCLAIMS high-fat diet (HFD), all at thermoneutrality (29 degrees C). Subsequently, mice were divided into 2 treatment groups: i) control group which remained under normoxia for 5 days, and ii) 5 days oxygen restriction to 13% ambient oxygen. On day 6, after 2 hour food removal at the start of the light phase, mice were killed immediately after taking them out of the indirect calorimetry system by decapitation. After sacrification, epididymal WAT was immediately dissected and snap frozen in liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from epididymal whiet adipose tissue using TRIZol Reagent (Sigma-Aldrich) according to the manufacturer’s instruction. After extraction, RNA was purified by using RNeasy columns (Qiagen). RNA concentration and purity were measured using the NanoDrop spectrophotometer (IsoGen Life Science). The integrity of RNA was checked with Experion automated electrophoresis system (BioRad).
|
| Label |
Cy5
|
| Label protocol |
1,000 ng of purified individual total RNA was used for cDNA synthesis, splitted in two equal fractions and used for subsequent cRNA labeling and synthesis using Cy5 and Cy3 dyes and the Agilent low RNA input fluorescent lineair amplification protocol, as described previously (Van Helden et al., Carcinogenesis 2010). Cy-3 labelled samples were pooled on an equimolar basis and served as reference pool; this comprised all samples present in the SuperSeries GSE53805.
|
| |
|
| Channel 2 |
| Source name |
reference pool
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
gender: male
age: adult
tissue: epididymal white adipose tissue
|
| Treatment protocol |
C57BL/6JOlaHsd wildtype male mice were acclimatized in the facility by feeding purified low fat diet (BIOCLAIMS, Hoevenaars et al., Genes and Nutrition 2012) for 3 weeks, followed by 12 weeks BIOCLAIMS high-fat diet (HFD), all at thermoneutrality (29 degrees C). Subsequently, mice were divided into 2 treatment groups: i) control group which remained under normoxia for 5 days, and ii) 5 days oxygen restriction to 13% ambient oxygen. On day 6, after 2 hour food removal at the start of the light phase, mice were killed immediately after taking them out of the indirect calorimetry system by decapitation. After sacrification, epididymal WAT was immediately dissected and snap frozen in liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from epididymal whiet adipose tissue using TRIZol Reagent (Sigma-Aldrich) according to the manufacturer’s instruction. After extraction, RNA was purified by using RNeasy columns (Qiagen). RNA concentration and purity were measured using the NanoDrop spectrophotometer (IsoGen Life Science). The integrity of RNA was checked with Experion automated electrophoresis system (BioRad).
|
| Label |
Cy3
|
| Label protocol |
1,000 ng of purified individual total RNA was used for cDNA synthesis, splitted in two equal fractions and used for subsequent cRNA labeling and synthesis using Cy5 and Cy3 dyes and the Agilent low RNA input fluorescent lineair amplification protocol, as described previously (Van Helden et al., Carcinogenesis 2010). Cy-3 labelled samples were pooled on an equimolar basis and served as reference pool; this comprised all samples present in the SuperSeries GSE53805.
|
| |
|
| |
| Hybridization protocol |
Individual Cy5-labelled samples were hybridized against the Cy3-labelled reference pool according to the manufacturers' procedure using Agilent In Situ Hybridization Kit Plus and GEx Hybridization Buffer HI-RPM. Samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers and hybridized at 65C for 17 hours at 10rpm rotation. After hybridization, slides were washed sequential following Agilents' recommendations and finally covered with Ozone-barrier slides.
|
| Scan protocol |
Scanned with an Agilent Technologies Scanner G2505B with 10 and 100% laser-power intensities.
|
| Description |
Cy3 samples were pooled on a equimolar basis and served as reference pool, and individual Cy5-labelled samples were hybridized against the reference pool.
|
| Data processing |
Images were quantified using Agilent Feature Extraction Software (v 10.7.3.1) to obtain raw median signal and background values for both Cy5 and Cy3. Spots with a mean signal higher than twice the background value over all arrays were considered to be expressed. Data was normalized according to Pellis et al., (Physiol Genomics 2003; 16:99-106) based on the Cy3-reference pool, and log2 transformation.
|
| |
|
| Submission date |
Jan 03, 2014 |
| Last update date |
Apr 06, 2017 |
| Contact name |
Evert M. van Schothorst |
| E-mail(s) |
evert.vanschothorst@wur.nl
|
| Organization name |
Wageningen University
|
| Lab |
Human and Animal Physiology
|
| Street address |
De Elst 1
|
| City |
Wageningen |
| ZIP/Postal code |
6708 WD |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL13912 |
| Series (3) |
| GSE53802 |
Hypoxia-induced metabolic dysfunction in WAT |
| GSE53804 |
Temperature-flux induced metabolic adjustments in WAT |
| GSE53805 |
External factors inducing metabolic adaptations in white adipose tissue in wildtype C57BL/6J mice housed at thermoneutrality. |
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