|
| Status |
Public on Jan 05, 2015 |
| Title |
Spleen female 2 |
| Sample type |
RNA |
| |
|
| Source name |
normal spleen
|
| Organism |
Rattus norvegicus |
| Characteristics |
tissue: spleen
Sex: female
strain: Han Wistar-derived rats (AlpkHsdBrlHan:WIST)
|
| Treatment protocol |
Rats were humanely killed at a designated establishment by halothane inhalation, which is an appropriate method under Schedule 1 of the Act.Organs and tissues were removed and cleaned of extraneous tissue by a 5-person necropsy team within 5 min of death, then frozen in liquid nitrogen. Pancreas was prioritized for rapid freezing, since its mRNA is known to be particularly vulnerable to degredation. Tissue samples from each animal weighing 100-250 mg were taken from frozen samples and homogenized immediately in RNeasy lysis buffer. Care was taken to ensure samples were taken consistently from the same region of tissue for each individual, and that they were representative of the whole organ (e.g. radial segments of kidney were taken from the equator in each case).
|
| Growth protocol |
Rats were housed 2-3 per cage
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified from lysates using Qiagen RNeasy mini columns according to the manufacturer’s instructions. Nucleic acid (RNA) purity was determined according to the 260/280nM absorbance ratio using a Nanodrop-1000 (Thermo Scientific). Samples were accepted only if the ratio fell between 1.8 and 2.2. Otherwise, RNA was re-extracted from new samples of frozen tissue. RNA integrity was assessed using the 2100 Bioanalyser (Agilent). Samples with an RNA integrity number (RIN) <7 were rejected, and re-processed.
|
| Label |
biotin
|
| Label protocol |
cDNA, biotin-labelled cRNA and fragmented hybridization probes were prepared using the Affymetrix IVT kit (Affymetrix CA.).
|
| |
|
| Hybridization protocol |
Hybridization mixes including control oligos and biotin-labelled cRNA were prepared following the Affymetrix protocol. Hybridisation to Affymetrix RAE230_2 rat genechips® was carried out overnight at 45oC rotating at 60 rpm. Bound biotinylated probes were revealed with strptavidin-phycoerythrin complex according to the Affymetrix protocol.
|
| Scan protocol |
Scanning was carried out with an Affymetric 3000 scanner using the RAE_230_V2 protocol
|
| Data processing |
R bioconductor was used for primary analysis, analysis was using GC-RMA-s
|
| |
|
| Submission date |
Nov 17, 2014 |
| Last update date |
Jan 05, 2015 |
| Contact name |
Russell Huby |
| Organization name |
AstraZeneca
|
| Street address |
Alderley Park
|
| City |
Macclesfield |
| State/province |
Cheshire |
| ZIP/Postal code |
SK10 4TG |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL1355 |
| Series (1) |
| GSE63362 |
Identification of sexually dimorphically expressed genes in rat tissues |
|
