|
| Status |
Public on Sep 21, 2012 |
| Title |
7th fraction of Ho series slicing |
| Sample type |
RNA |
| |
|
| Source name |
5 micron x 200 horizontal a little oblique slices (1 mm width) of the brain, set 2
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
tissue: brain
gender: male
age: 8 weeks
section type: horizontal oblique plane
mouse id: 6
|
| Growth protocol |
Mice were grown in pathogen-free conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the Mag-max-96 for Microarrays Total RNA Isolation Kit (Ambion) following the manufacturer's recommendations of the spin column protocol. 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 RNeasy 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 manufacturer's instructions. On completion of the fragmentation reaction, 250 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays 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 using the 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 |
Ho_07
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Data were normalized using per chip: normalize to 50th percentile.
|
| |
|
| Submission date |
Mar 09, 2012 |
| Last update date |
Sep 21, 2012 |
| Contact name |
Yuko Okamura-Oho |
| E-mail(s) |
yoho-tky@umin.ac.jp
|
| Organization name |
RIKEN
|
| Street address |
2-1, Hirosawa
|
| City |
Wako-shi |
| State/province |
Saitama |
| ZIP/Postal code |
351-0198 |
| Country |
Japan |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE36408 |
Gene expression profiling of the whole mouse brain using transcriptome tomography |
|
