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Status |
Public on Jun 30, 2011 |
Title |
Mus Musculus_wild-type fully-grown oocyte_rep2 |
Sample type |
RNA |
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Source name |
Mus musculus oocyte wild-type
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Organism |
Mus musculus |
Characteristics |
gender: Female age: 8-12 weeks-old mice cell type: Oocyte developmental stage: Fully-grown, GV germinal vesicule stage genetic background: wild-type genotype: mixed 129XI/SvJ and C57BL/6
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Growth protocol |
Fully grown oocytes (GV) were collected from ovaries of 8-12 week-old mice (WT, Hsf1 mutant or Hsf2 mutant) in M2 medium (SIGMA).
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA from 200 to 400 fully grown oocytes (GV) from each genotype (WT, Hsf1 mutant or Hsf2 mutant) were extracted using Qiagen RNeasy micro kit and 10ng samples of RNA were amplified using NuGen WT ovation Pico RNA amplification system to obtain about 10µg of single-strand (ss) cDNA. Two micrograms of ss cDNA was converted to double-strand cDNA using Klenow fragment of the DNA polymerase I and the synthesized cDNAs were then incubated with 1μl of 4 mg/ml RNase A at 37 °C for 10 minutes, precipitated and the pellet dissolved with 20μl of nuclease-free water.
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Label |
Cy5
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Label protocol |
The cDNA of each sample (3.5μg) was synthesized as probe and labeled with Cy3- or Cy5-conjugated random nonamers (TriLink Biotechnologies, San Diego, CA).
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Hybridization protocol |
Labelled cDNA were hybridized to a NimbleGen Mus musculus Gene Expression 385K microarray, containing 42,586 probe sets with up to 9 probes of 60mer oligonucleotides per gene, following the protocol by Roche NimbleGen, Inc. (Madison, USA). The microarrays were incubated on the NimbleGen Hybridization System 4 (Roche NimbleGen) for 16 hours at 42 °C. The hybridized slides were washed at 10× wash buffer I, II and III (Roche NimbleGen) and dried by nitrogen gas at room temperature.
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Scan protocol |
Slides were scanned with an Axon GenePix Pro 4200A microarray scanner at 5μm resolution, 532 nm and 635nm wavelengths software using associated software GenePix Pro software (Molecular Devices, Sunnyvale, CA). The scanned images of the arrays were quantified using NimbleScan software (Roche NimbleGen).
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Description |
This sample is of wild-type oocytes. It is the second of two wild-type technical replicates used in this experiment.
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Data processing |
The raw data (.pair file) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction as implemented in the NimbleScan software package, version 2.4.27 (Roche NimbleGen, Inc.).
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Submission date |
Dec 21, 2010 |
Last update date |
Jun 30, 2011 |
Contact name |
Florent LE MASSON |
E-mail(s) |
lemasson@cict.fr
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Phone |
0561557750
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Organization name |
CNRS
|
Lab |
Centre de Biologie du Développement
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Street address |
118 route de Narbonne Bat4R3b3
|
City |
Toulouse |
ZIP/Postal code |
31062 |
Country |
France |
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Platform ID |
GPL7437 |
Series (1) |
GSE26240 |
Expression analysis of fully-grown oocyte collected from mouse wild-type, Hsf1 mutant or Hsf2 mutant |
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