|
| Status |
Public on Aug 27, 2013 |
| Title |
LFD, 5days, replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
epididymal white adipose tissue, LFD, 5 days
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
genotype: wildtype
gender: male
developmental stage: adult
tissue: epididymal white adipose
treatment: low-fat diet
treatment duration: 5 days
|
| Treatment protocol |
Animals were fed a purified high-fat diet (HDF) or low-fat diet (LFD); diets are published elsewhere (Hoevenaars et al., Genes Nutrition 2012 (PMID 22228221)). All groups received the diet ad-libitum during 5 days or 12 weeks intervention. Animals were fasted for 2 hr prior to anaesthetization and sacrification.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from epididymal white adipose tissue using TRIZol Reagent (Sigma-Aldrich) according to the manufacturer's instructions. 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 the Experion automated electrophoresis system (BioRad).
|
| Label |
Cy5
|
| Label protocol |
1,000 ng of purified individual total RNA was used for cDNA synthesis. The cDNA was split into two equal fractions and used for subsequent cRNA labeling and synthesis using Cy5 and Cy3 dyes and the Agilent Low RNA Input Fluorescent Linear Amplification protocol, as described previously (Van Helden et al., Carcinogenesis 2010 (PMID 20472610)).
|
| |
|
| Channel 2 |
| Source name |
Reference pool of all samples
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
genotype: wildtype
gender: male
developmental stage: adult
tissue: epididymal white adipose
sample type: reference
|
| Treatment protocol |
Animals were fed a purified high-fat diet (HDF) or low-fat diet (LFD); diets are published elsewhere (Hoevenaars et al., Genes Nutrition 2012 (PMID 22228221)). All groups received the diet ad-libitum during 5 days or 12 weeks intervention. Animals were fasted for 2 hr prior to anaesthetization and sacrification.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from epididymal white adipose tissue using TRIZol Reagent (Sigma-Aldrich) according to the manufacturer's instructions. 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 the Experion automated electrophoresis system (BioRad).
|
| Label |
Cy3
|
| Label protocol |
1,000 ng of purified individual total RNA was used for cDNA synthesis. The cDNA was split into two equal fractions and used for subsequent cRNA labeling and synthesis using Cy5 and Cy3 dyes and the Agilent Low RNA Input Fluorescent Linear Amplification protocol, as described previously (Van Helden et al., Carcinogenesis 2010 (PMID 20472610)).
|
| |
|
| |
| Hybridization protocol |
Individual Cy5-labelled samples were hybridized against the Cy3-labelled reference pool according to the manufacturer's procedure using the 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 sequentially following Agilent's 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 |
LFD_5d 2
Cy3 samples were pooled on an equimolar basis and served as the reference pool, and individual Cy5-labelled samples were hybridized against the reference pool.
|
| Data processing |
Images were quantified using Agilent Feature Extraction Software (v 10.5.5.1) 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 (PMID 14570982)) based on the Cy3-reference pool, and log2 transformed.
|
| |
|
| Submission date |
May 30, 2012 |
| Last update date |
Aug 27, 2013 |
| Contact name |
Evert M. van Schothorst |
| E-mail(s) |
[email protected]
|
| 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 |
GPL10333 |
| Series (1) |
| GSE38337 |
Short-term, high fat-feeding-induced changes in white adipose tissue gene expression are highly predictive for long-term changes |
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