|
| Status |
Public on Jul 30, 2014 |
| Title |
cHF+Phospholipids+Rosi, replicate 7 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
cHF+Phospholipids+Rosi, replicate 7
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6N
gender: male
age: adult
tissue: liver
|
| Treatment protocol |
Animals were fed a control corn oil-based high-fat diet (cHF), or one of the cHF supplementations: 1) ~7 % of dietary lipids was replaced by the EPA and DHA concentrate in the form of omega-3 phospholipids rich in phosphatidylcholine (PC); 2) low dose rosiglitazone (R); and 3) both PC and rosiglitazone (PC+R). All groups received the diet ad-libitum during 7 weeks intervention.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from liver tissue stored in RNAlater Solution 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).
|
| |
|
| Channel 2 |
| Source name |
reference pool
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6N
gender: male
age: adult
tissue: liver
|
| Treatment protocol |
Animals were fed a control corn oil-based high-fat diet (cHF), or one of the cHF supplementations: 1) ~7 % of dietary lipids was replaced by the EPA and DHA concentrate in the form of omega-3 phospholipids rich in phosphatidylcholine (PC); 2) low dose rosiglitazone (R); and 3) both PC and rosiglitazone (PC+R). All groups received the diet ad-libitum during 7 weeks intervention.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from liver tissue stored in RNAlater Solution 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).
|
| |
|
| |
| 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 10.5.1.1) 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 |
Mar 18, 2013 |
| Last update date |
Jul 30, 2014 |
| 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) |
| GSE45235 |
Marine omega-3 phospholipids suppress hepatic steatosis by a complex inhibition of biosynthetic pathways in dietary obese mice |
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