|
| Status |
Public on Jan 31, 2020 |
| Title |
Liver_control_rep4 |
| Sample type |
RNA |
| |
|
| Source name |
Liver, control
|
| Organism |
Mus spretus |
| Characteristics |
strain/background: SPRET/EiJ
gender: Male
tissue: Liver
treatment: normal (Teklad) diet
age: 22 months
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the AllPrep DNA/RNA/Protein Mini Kit (QIAGEN) following the manufacturer's recommendations. The protocol includes an on-column DNase digestion. Total RNA was quantified in a NanoDrop ND-1000 Spectrophotometer and quality was confirmed by RNA 6000 Nano Bioanalyzer (Agilent Technologies, Palo Alto, California USA) assay.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 2000 ng RNA using the Agilent Low-Input QuickAmp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 µL containing 1x Fragmentation Buffer (Agilent) and 2x Gene Expression Blocking Agent (Agilent) following the manufacturer’s instructions. On completion of the fragmentation reaction, 55 µL of 2x Hi-RPM Hybridization Buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Mouse Gene Expression v2 8 x 60K Microarray (G4858A-074809) for 17 hours at 65°C in a rotating Agilent G2545A hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with Gene Expression Wash Buffer 1 (Agilent) and 1 minute with 37°C Gene Expression Wash Buffer 2 (Agilent), then dried in an ozone-free atmosphere.
|
| Scan protocol |
Microarray slides were scanned with an Agilent Microarray Scanner (Agilent G2565C) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3 µm, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
H4
Gene expression after 60 days of normal diet.
Replicate 4
|
| Data processing |
Image analysis and quantification of fluorescence data were carried out with Agilent Feature Extraction Software 11.5.1.1 using the Agilent protocol GE1_1105_Oct12, grid template 074809_D_F_20150624 and the QC Metric Set GE1_QCMT_Oct12, default parameters.
After background subtraction by RMA, quantile normalization was performed on pre-processed data with limma.
|
| |
|
| Submission date |
Jan 16, 2018 |
| Last update date |
Jan 31, 2020 |
| Contact name |
Nieves Abril |
| E-mail(s) |
bb1abdim@uco.es
|
| Phone |
957218139
|
| Organization name |
University of Córdoba
|
| Street address |
Campus Rabanales, edificio C6, 2ª planta
|
| City |
Cordoba |
| State/province |
Not Applicable |
| ZIP/Postal code |
14071 |
| Country |
Spain |
| |
|
| Platform ID |
GPL21810 |
| Series (1) |
| GSE109254 |
Transcriptomic modifications associated to protective effect of Pedro-Ximenez must on p,p´- DDE-induced liver injury in aged Mus spretus mice |
|
