|
| Status |
Public on Jul 31, 2013 |
| Title |
Br-P25-rep4 |
| Sample type |
RNA |
| |
|
| Source name |
whole brain-tissue-P25-replicate4
|
| Organism |
Rattus norvegicus |
| Characteristics |
tissue: whole brain
age: P25
|
| Growth protocol |
rats were deeply anestesized with 7% chloralhydrate (1ml/100g bodyweight), Tissue preparation was carried out in chilled buffer solution (~4 °C) containing (mM): 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, 260 D-glucose, 2 sodium pyruvate, 3 myo-inositol and 1 kynurenic acid. The solution was gassed 30 min with 95% O2 and 5% CO2 to adjust the pH to 7.4. The brainstem was dissected and coronal slices (300-µm thickness) containing the SOC were cut with a vibratome. The SOC was dissected and collected. Whole brain and SOC tissue was stored in RNAlater (Ambion) at -80°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA used for hybridization experiments was isolated from one entire brain, or from the SOCs of single animals at P16 and P25; the SOCs from 4 animals were pooled at P0 and from 2 animals at P4. Total RNA extraction from SOC tissue was performed with the RNeasy Lipid Tissue Kit (Qiagen), from the whole brain with the RNeasy Midi Kit (Qiagen), both according to the manufacturers instructions.
|
| Label |
Cy3
|
| Label protocol |
The synthesis of the cRNA samples (Cy3 or Cy5) was performed according to the manufacturers protocol with the Agilent Low RNA Input Linear Amplification Kit. 1,000 ng total RNA were used as starting material. The cRNA samples were purified by RNAeasy columns (QIAGEN). The incorporation rate was measured with a Nanodrop ND-1000 UV-Vis Spectrophotometer (specific activity > 9.0 pmol Cy3 or Cy5/µg cRNA).
|
| |
|
| Hybridization protocol |
825ng of Cy3-labelled and 825ng of Cy5-labelled cRNA were mixed and fragmented at 60°C for 30 minutes in a reaction volume of 55 µl containing 25x Agilent fragmentation buffer and 11 µl 10x Agilent blocking agent. 55 µl GEx hybridization buffer HI-RMP was added to the fragmentation mixture and 100 µl were hybridized to Agilent Whole Rat Genome Oligo Microarrays (G4131F, 4x44k format) for 17 hours at 65°C (10 rpm). After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) followed by 1 min with 37°C prewarmed GE Wash Buffer 2 (Agilent).
|
| Scan protocol |
Immediately after the washing steps, the microarrays were scanned with an The Agilent DNA microarray Scanner (48-slide scanning system). The settings were: scan area 61x21.6 mm, Scan resolution 5µm, Dye channel Red & Green, PMT for both channels XDR Hi 100% and XDR Lo 10%.
|
| Description |
none
|
| Data processing |
Raw data analysis was performed with Agilent Feature Extraction Software (v8.1) and data normalization and statistics by in-house software packages and algorithms of the Fraunhofer ITWM, Kaiserslautern. This implied a Lowess transformation with smoothing parameter of 0.2 to reduce intensity dependent errors. To identify differentially expressed genes, the non-parametric Fisher-Pitman-Test was used.
|
| |
|
| Submission date |
Jun 23, 2009 |
| Last update date |
Jul 31, 2013 |
| Contact name |
Hans Gerd Nothwang |
| E-mail(s) |
hans.g.nothwang@uni-oldenburg.de
|
| Phone |
0441-7983932
|
| Organization name |
University of Oldenburg
|
| Department |
Neurogenetics
|
| Street address |
Carl von Ossietzky Str. 9-11
|
| City |
Oldenburg |
| State/province |
Lower Saxony |
| ZIP/Postal code |
26129 Oldenburg |
| Country |
Germany |
| |
|
| Platform ID |
GPL7294 |
| Series (3) |
| GSE16764 |
Comparative microarray analyses between the rat superior olivary complex and the brain |
| GSE34003 |
Time course expression study in the rat superior olivary complex |
| GSE36470 |
Rat superior olivary complex |
|
