|
| Status |
Public on Oct 07, 2014 |
| Title |
E9.5 trunk, Arid3b-null, replicate2 |
| Sample type |
RNA |
| |
|
| Source name |
E9.5 trunk, Arid3b-null, replicate2
|
| Organism |
Mus musculus |
| Characteristics |
developmental stage: E9.5
tissue: trunk
phenotype: Arid3b-null
|
| Extracted molecule |
total RNA |
| Extraction protocol |
E9.5 embryos were dissected in cold PBS. The trunk was isolated (including the body from the otic vesicle to somite 15 and forelimbs). Four wild-type and four mutant embryos were collected and pooled for each of the microarray triplicates. RNA was extracted with the Qiagen extraction kit and RNA quality was checked by Nanodrop spectrophotometer measure and gel electrophoresis.
|
| Label |
Cy3
|
| Label protocol |
The One-Color Microarray-Based Gene Expression Analysis Protocol (Agilent Technologies, Palo Alto, CA, USA) was used to amplify and label RNA. Briefly, 200-800 ng of total RNA was reverse transcribed using T7 promoter Primer and MMLV-RT. Then cDNA was then converted to aRNA using T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3-labeled CTP.
|
| |
|
| Hybridization protocol |
Samples were hybridised to Whole Mouse Genome Microarray 4 x 44K ( G4846A, Agilent Technologies). 1.65 µg of Cy3 labelled aRNA were hybridised for 17 hours at 65ºC in a hybridisation oven (G2545A, Agilent) set to 10 rpm in a final concentration of 1X GEx Hybridization Buffer HI-RPM, according to the manufacturer's instructions (One-Color Microarray-Based Gene Expression Analysis, Agilent Technologies).
|
| Scan protocol |
Arrays were scanned at 5µm resolution on an Agilent DNA Microarray Scanner (G2565BA, Agilent Technologies) using the default settings for 4x44k format one-color arrays. Images provided by the scanner were analysed using Feature Extraction software version 9.5.3.1 (Agilent Technologies).
|
| Description |
Gene expression in E9.5 trunks
|
| Data processing |
Data were filtered at a probe level according to the following criteria. We demanded that at least 75% of the probes in at least one experimental condition had a quantification flag denoting that the signal is distinguishable from background. The same criterion is applied independently for the saturation of the signal. In addition, we filtered out those probes that had more than 25% of the replicates in at least one experimental condition with a flag indicating presence of outliers in the quantification of the signal probe.
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| |
|
| Submission date |
Oct 06, 2014 |
| Last update date |
Oct 07, 2014 |
| Contact name |
Juan Jose Sanz-Ezquerro |
| E-mail(s) |
[email protected]
|
| Phone |
+34915855395
|
| Organization name |
Centro Nacional dde Biotecnologia
|
| Department |
Biologia Molecular y Celular
|
| Street address |
Darwin, 3
|
| City |
Madrid |
| ZIP/Postal code |
28049 |
| Country |
Spain |
| |
|
| Platform ID |
GPL11202 |
| Series (2) |
| GSE62071 |
Gene expression in E9.5 trunks comparing wt with Arid3b-null embryos |
| GSE62072 |
Arid3b is required for heart development |
|
