|
| Status |
Public on Jun 21, 2013 |
| Title |
Liver_dye_swap_experiment |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Liver, Rap1 knockout mouse -/-, 30 weeks
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL6/129SV (90%: 10%)
genotype/variation: Rap1 -/-
tissue: liver
age: 30 weeks
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with Trizol. The QIAGEN kit was employed for purification. RNA Integrity Numbers were in the range 6.6 to 8.7 when assayed by Lab-chip technology on an Agilent 2100 Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Amount of nucleic acid labeled: 100ng. Commercial Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) version 6.5 kit by following manufacturer instructions. Agilent manual G4140-90050 of May 2010. Amplification: by RNA polymerases. Briefly, MMLV-RT retrotranscription of sample from a T7 promoter primer is followed by a T7 RNA pol catalysed in vitro transcription reaction in the presence of either Cy3-CTP or Cy5-CTP fluorophores. Labeled samples are purified with silica-based RNeasy spin columns (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
Liver, wild-type mouse, 30 weeks
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL6/129SV (90%: 10%)
genotype/variation: wild-type
tissue: liver
age: 30 weeks
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with Trizol. The QIAGEN kit was employed for purification. RNA Integrity Numbers were in the range 6.6 to 8.7 when assayed by Lab-chip technology on an Agilent 2100 Bioanalyzer.
|
| Label |
Cy5
|
| Label protocol |
Amount of nucleic acid labeled: 100ng. Commercial Two-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) version 6.5 kit by following manufacturer instructions. Agilent manual G4140-90050 of May 2010. Amplification: by RNA polymerases. Briefly, MMLV-RT retrotranscription of sample from a T7 promoter primer is followed by a T7 RNA pol catalysed in vitro transcription reaction in the presence of either Cy3-CTP or Cy5-CTP fluorophores. Labeled samples are purified with silica-based RNeasy spin columns (Qiagen).
|
| |
|
| |
| Hybridization protocol |
Hybridization chamber type: SureHyb hybridization chamber (Agilent). Quantity of each labeled extract used: 300 ng. Volume: 50 uL. Temperature (ºC): 65. Duration: 17 hours.
|
| Scan protocol |
Scanned on a G2505C DNA microarray scanner (Agilent). Images were quantified using Agilent Feature Extraction Software (ver. 10.7).
|
| Description |
LIVER_DYE-SWAP_1
Dye-swap experiment.
|
| Data processing |
Microarray background subtraction was carried out using normexp method. Normalization was performed using lowess for within array normalization and quantiles for between array normalization. Differentially expressed genes were obtained by applying linear models with R limma package (Smyth, 2005) (Bioconductor project, http://www.bioconductor.org). To account for multiple hypotheses testing, the estimated significance level (p-value) was adjusted using Benjamini & Hochberg False Discovery Rate (FDR) correction.
|
| |
|
| Submission date |
Dec 27, 2012 |
| Last update date |
Jun 21, 2013 |
| Contact name |
Paula Martinez |
| E-mail(s) |
pmartinez@cnio.es
|
| Organization name |
CNIO
|
| Street address |
Melchor Fernandez Almagro 3
|
| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
| |
|
| Platform ID |
GPL10787 |
| Series (2) |
| GSE43173 |
RAP1 protects from obesity, diabetes and metabolic syndrome through its extra telomeric role regulating gene expression (old mouse liver) |
| GSE43174 |
RAP1 protects from obesity, diabetes and metabolic syndrome through its extra telomeric role regulating gene expression |
|
