|
| Status |
Public on May 21, 2015 |
| Title |
Repl.3_Area17 Outer Subventricular Zone |
| Sample type |
RNA |
| |
|
| Source name |
Repl.3_Area17 Outer Subventricular Zone
|
| Organism |
Mustela putorius furo |
| Characteristics |
area: 17
zone: outer subventricular
tissue: Cerebral cortex
age: Postnatal day 2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the RNeasy Mini RNA Purification kit (QUIAGEN, Cat. No. 74104) following the manufacturer's recommendations. The protocol includes cell lysis and an on-column DNase digestion. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent-036832 Ferret_44K_v3 for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
3A17OSVZ
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Aug 22, 2014 |
| Last update date |
May 21, 2015 |
| Contact name |
Victor Borrell |
| E-mail(s) |
vborrell@umh.es
|
| Organization name |
CSIC
|
| Department |
Institute of Neuroscience
|
| Lab |
221
|
| Street address |
Av Ramon y Cajal s/n
|
| City |
San Juan de Alicante |
| State/province |
Alicante |
| ZIP/Postal code |
03550 |
| Country |
Spain |
| |
|
| Platform ID |
GPL19111 |
| Series (1) |
| GSE60687 |
Sharp changes in gene expression levels along germinal layers distinguish the development of gyrencephaly |
|
