|
| Status |
Public on Feb 23, 2014 |
| Title |
3_RNA from posterior half of wild-type embryo |
| Sample type |
RNA |
| |
|
| Source name |
embryo_wild-type
|
| Organism |
Mus musculus |
| Characteristics |
genotype/variation: wild-type
developmental stage: E9.5
tissue: posterior half of embryo
|
| Treatment protocol |
Embryos were collected at E9.5 and posterior half of embryos were frozen immediately at -80°C.
|
| Growth protocol |
Jmjd3+/- mice were intercrossed and embryos were collected at E9.5 according to the time after mating and the somite number.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After genotyping, total RNAs were extracted using RNeasy mini RNA purification kit (QIAGEN, Valencia, CA). RNA was quantified 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 0.4 ug RNA using the Low RNA Input Fluorescent 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 |
1.65 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 55 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Mouse Whole Genome Microarray 4x44K (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 by raise it slowly from buffer 2.
|
| 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 5um, Dye channel is set to Green. Scan mode is eXtended Dynamic range Scan mode and PMT is set to 10% and 100%).
|
| Description |
Gene Expression of posterior half of wild-type embryo
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 014868_D_F_20100617) to obtain background subtracted and spatially detrended Processed Signal intensities. These data were analyzed further with the GeneSpring GX11.5 software (Agilent Technologies). Normalization was performed as follows: 1, intensity-dependent Lowess normalization; 2, data transformation, with measurements less than 0.01 set to 0.01; 3, per-chip normalization, in which the 75th percentile method was used to normalize each array; 4, per-gene normalization, in which the data were normalized to control samples (wild-type).
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| |
|
| Submission date |
Aug 23, 2012 |
| Last update date |
Feb 23, 2014 |
| Contact name |
Takumi Nishiuchi |
| E-mail(s) |
tnish9@staff.kanazawa-u.ac.jp
|
| Organization name |
Kanazawa University
|
| Street address |
13-1 Takaramachi
|
| City |
Kanazawa |
| ZIP/Postal code |
920-0934 |
| Country |
Japan |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE40332 |
Gene expression profiles in wild-type and Jmjd3-/- embryos |
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