|
| Status |
Public on Sep 07, 2012 |
| Title |
cHF+ROSI, replicate 5, part 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Gastrocnemius muscle, high fat diet + rosiglitazone (10mg/kg diet), sacrificed after 'diet-switch protocol'
|
| Organism |
Mus musculus |
| Characteristics |
treatment: cHF diet supplemented with rosiglitazone
tissue: gastrocnemius muscle
strain: C57BL/6N
gender: male
age: adult
|
| Treatment protocol |
Animals were fed a purified high-fat diet as a control (cHF), or either supplemented with long chain n-3 PUFA (cHF+F), or rosiglitazone (10 mg/kg diet; cHF+ROSI). An additional group received cHF+ROSI with a higher dose of rosiglitazone 100 mg/kg diet). All groups received the diet ad-libitum during the 8 weeks intervention. Animals were fasted during 10hr in the light phase, and re-fed Chow diet during the subsequent night. The next morning, animals were anaestetized and sacrificed. The combination treatment group receiving both LC n-3 PUFA and low dose rosiglitazone (cHF+F+ROSI) is presented in another GEO study, GSE36717.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from gastrocnemius muscle stored in RNAlater Solution (Ambion) using TRI 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). Here, the additional group receiving cHF+ROSI at 100 mg/kg diet was not included in normalization and data analysis, so these arrays are therefore lacking in the list. Samples were nevertheless included for the Cy3 labelling and therefore included in the (Cy3) reference pool.
|
| |
|
| Channel 2 |
| Source name |
reference pool (all 2 diets)
|
| Organism |
Mus musculus |
| Characteristics |
treatment: reference pool (all 2 diets)
tissue: gastrocnemius muscle
strain: C57BL/6N
gender: male
age: adult
|
| Treatment protocol |
Animals were fed a purified high-fat diet as a control (cHF), or either supplemented with long chain n-3 PUFA (cHF+F), or rosiglitazone (10 mg/kg diet; cHF+ROSI). An additional group received cHF+ROSI with a higher dose of rosiglitazone 100 mg/kg diet). All groups received the diet ad-libitum during the 8 weeks intervention. Animals were fasted during 10hr in the light phase, and re-fed Chow diet during the subsequent night. The next morning, animals were anaestetized and sacrificed. The combination treatment group receiving both LC n-3 PUFA and low dose rosiglitazone (cHF+F+ROSI) is presented in another GEO study, GSE36717.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from gastrocnemius muscle stored in RNAlater Solution (Ambion) using TRI 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). Here, the additional group receiving cHF+ROSI at 100 mg/kg diet was not included in normalization and data analysis, so these arrays are therefore lacking in the list. Samples were nevertheless included for the Cy3 labelling and therefore included in the (Cy3) reference pool.
|
| |
|
| |
| 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 using a Cy5-dyesaver as published (Anal Biochem 2007_363_315).
|
| 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 10.5.5.1) 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) based on the Cy3-reference pool, and log2 transformation.
series suplementary file contains normalized log2 value of sample over reference pool (minus bad spots)
|
| |
|
| Submission date |
Mar 22, 2012 |
| Last update date |
Sep 07, 2012 |
| 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 |
GPL4134 |
| Series (2) |
| GSE36716 |
Muscle Involvement in Preservation of Metabolic Flexibility by Treatment using n-3 PUFA or Rosiglitazone in Dietary-Obese Mice |
| GSE36718 |
Muscle Involvement in Preservation of Metabolic Flexibility by Treatment using n-3 PUFA, Rosiglitazone, and a combination of both |
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