|
| Status |
Public on Apr 15, 2014 |
| Title |
Oocyte_prepubertal_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Oocyte, prepubertal B6C3F1 mice, 3 weeks of age, replicate 2
|
| Organism |
Mus musculus |
| Characteristics |
strain: B6C3F1
cell type: oocyte
age: 3 weeks of age
developmental stage: prepubertal
|
| Treatment protocol |
Mice were injected with 10 IU PMSG and 10 IU hCG i.p. 48 hours apart, and sacrificed by cervical dislocation 14 h after hCG injection. Metaphase II oocytes were retrieved from the oviductal ampullae, and freed of cumulus cells and zonae pellucidae, before lysing in SDS lysis buffer (4% SDS, 100 mM Tris/HCl pH 7.5, 0.1 M DTT, total volume 70 µL).
|
| Growth protocol |
B6C3F1 mice (C57Bl/6J · C3H/HeN) aged 3, 8 and 52 weeks (prepubertal, mid-age, climacteric) were maintained and used for experiments according to the ethical permit issued by the Landesamt für Natur, Umwelt und Verbraucherschutz (LANUV) of the state of North Rhine-Westphalia, Germany. Throughout the extended time of this study, mice were reared, housed, fed ad libitum and primed for superovulation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA (samples in 300 μL RLT buffer with 1% β-mercapt- oethanol) was isolated using RNeasy Micro Kit as described by the manufacturer (Qiagen, no DNase treatment). An examina- tion of total RNA on an Agilent 2100 Bioanalyzer and RNA Pico 6000 Lab-Chip kit confirmed the extraction of high-quality RNA, which was then prepared for gene expression profiling. Arcturus RiboAmp HS Plus Amplification Kit (MDS Analytical Technologies GmbH, Germany) was used to amplify total RNA (two rounds of amplification) according to manufacturer’s instructions. After the second round of amplification, ampli- fied RNA was eluted with 15 μL RE buffer. Concentration of amplified RNA was measured using Agilent Bioanalyzer 2100 and RNA 6000 Lab-Chip kit.
|
| Label |
Cy3
|
| Label protocol |
Three micrograms of amplified RNA were labeled with Cy3 using Arcturus Turbo Labeling CY3 Kit (MDS Analytical Technologies GmbH, Germany). Concentration and frequency of incorporation (FOI) were measured using the NanoPhotometer (Implen, Munich, Germany).
|
| |
|
| Hybridization protocol |
Fragmentation (1650 ng Cy3-labeled amplified RNA) and hybridization were performed following the hybridization pro- cedure recommended by the array manufacturer (Agilent Tech- nologies), with a modification given by the manufacturer of the Turbo Labeling CY3 Kit.
|
| Scan protocol |
Microarray wash and detection of the labeled RNA on GeneChips were performed according to the instructions of Agilent Technologies. Gene expression profiling was performed using Agilent’s Whole Mouse Genome Oligo Microarrays (4 44k, each array with 41 174 features).
|
| Description |
Gene expression of prepubertal mouse oocyte
|
| Data processing |
Array image acquisition and feature extraction was performed using the Agilent G2505B Microarray Scanner and Feature Extraction software version 9.5 (Agilent Technologies).
|
| |
|
| Submission date |
Dec 17, 2012 |
| Last update date |
Apr 15, 2014 |
| Contact name |
Marcin Siatkowski |
| E-mail(s) |
[email protected]
|
| Phone |
0048668859148
|
| Organization name |
IBIMA
|
| Street address |
Ernst-Heydemann-Str. 8
|
| City |
Rostock |
| ZIP/Postal code |
18059 |
| Country |
Germany |
| |
|
| Platform ID |
GPL11202 |
| Series (1) |
| GSE42959 |
Maternal age effect on mouse oocytes: new biological insight from proteomic analysis |
|
