|
| Status |
Public on Apr 01, 2012 |
| Title |
E14.5 Nanos2 Hetero 2 |
| Sample type |
RNA |
| |
|
| Source name |
embryonic male gonad
|
| Organism |
Mus musculus |
| Characteristics |
tissue: gonad
Sex: male
age: E14.5
genotype: Nanos2+/-
genetic background: mixed ICR and N2
|
| Treatment protocol |
Embryonic male gonads were disected in ice-cold PBS and were immeadiately frozen in RNAlater (Ambion). Frozen tissues were stored at -80 degree until total RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with RNeasy mini kit (QIAGEN, Valencia, CA) according to the manufacture's protocol.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.20 µg RNA using the One-Color Low RNA Input Linear Amplification 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 50 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 50 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo Microarrays (G4122F) 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.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression E14.5 mouse male gonad Nanos2 hetero
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5 (Agilent) using default parameters (protocol GE1-v5_95 and Grid:014868_D_20070207) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Oct 21, 2011 |
| Last update date |
Apr 01, 2012 |
| Contact name |
Rie Saba |
| Organization name |
National Institute of Genetics
|
| Department |
Genetic Strain Research Center
|
| Lab |
Mammalian Development
|
| Street address |
Yata 1111
|
| City |
Mishima |
| State/province |
Shizuoka |
| ZIP/Postal code |
411-8540 |
| Country |
Japan |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE33138 |
Gene expression of Tg 3×FLAG-Nanos2-deltaN10 mouse male gonad |
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