|
| Status |
Public on Dec 23, 2013 |
| Title |
Cerebellum_rep3 En2KO |
| Sample type |
RNA |
| |
|
| Source name |
entire cerebellum
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Cerebellum
genotype: En2KO
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from fresh tissue using RNAeasy miniki (Qiagen) following the manufacturer's recommendations. The protocol includes 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 1 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 Whole Human Genome Oligo Microarrays (G4112A) 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 |
The original *En2* mutants (mixed 129Sv x C57BL/6 and outbred genetic background) were crossed at least five times into a C57BL/6 background.
|
| Data processing |
The scanned images were analyzed with FeatureExtractor (protocol GE1_107_Sep09 and Grid: 014868_D_F_20120131) . Intensity values were then processed with Agi4x44PreProcess using default parameters to remove low-quality probes. Signals where then normalized by means of the quantile normalization method.
|
| |
|
| Submission date |
Oct 23, 2013 |
| Last update date |
Dec 23, 2013 |
| Contact name |
Erik Dassi |
| E-mail(s) |
erik.dassi@unitn.it
|
| Organization name |
University of Trento
|
| Department |
CIBIO
|
| Street address |
Via Sommarive, 9
|
| City |
Trento |
| State/province |
TN |
| ZIP/Postal code |
38123 |
| Country |
Italy |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE51612 |
Transcriptome profiling in Engrailed2 knockout mice reveals common molecular pathways associated with ASD. |
|
