|
| Status |
Public on Jan 20, 2010 |
| Title |
Biological replicate 3 - epi vs mes (before vs after EMT) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
epithelial OSE in 2.5% FBS, before EMT
|
| Organism |
Mus musculus |
| Characteristics |
cell type: OSE cells
|
| Treatment protocol |
To maintain the epithelial phenotype, the cells were cultured in 2.5% FBS. To induce the mesenchymal phenotype, they were cultured in 15% FBS for one week.
|
| Growth protocol |
Superovulation was induced in virgin female C57/Bl6 mice with the following scheme: day 0: administration of Pregnant mare's serum gonadotropin (PMSG); day 1: administration of hCG and mating with stud male; day 3: female culled 12 hours post-coitum and ovaries collected. The external capsula was removed, the ovaries were dissected and the cells were dissociated by treating them for 10 minutes in collagenase + dispase, then 2 X 10 minutes in trypsin. The OSE cells were then cultured in 25% DMEM, 25% F12, 25% MCDB-105, 25% 199 medium in the absence of antibiotics.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cells at 80% confluence were collected by cell scraping in ice cold PBS, spun down for 5 minutes at 1000 rpm at 4deg, snap frozen and stored at -80deg. Total RNA was extracted using the Macherey-Nagel RNA L Kit, following the manufacturer's recommendations, which includes a DNAseI treatment step. Elution was made in RNAse-free wtaer. The RNA quality was checked using the Agilent 2100 bioanalyzer. The RNA concentration was measured using the Vysis Nanodrop.
|
| Label |
Cy3,Cy5
|
| Label protocol |
The amplification of the extracted RNA was made using the Amino Allyl MessageAmp aRNA kit (Ambion, 1752), following the manufactirer's instructions, starting from 4ug of total RNA per sample. The labelling was made using Cy3 Mono-Reactive Dye (Amersham, PA23001) and Cy5 Mono-Reactive Dye (Amersham, PA25001). RNA and Dye concentrations for each sample were determined using the Vysis Nanodrop.
|
| |
|
| Channel 2 |
| Source name |
mesenchymal OSEcultured in 15% FBS for one week (I.e. after EMT)
|
| Organism |
Mus musculus |
| Characteristics |
cell type: OSE cells
|
| Treatment protocol |
To maintain the epithelial phenotype, the cells were cultured in 2.5% FBS. To induce the mesenchymal phenotype, they were cultured in 15% FBS for one week.
|
| Growth protocol |
Superovulation was induced in virgin female C57/Bl6 mice with the following scheme: day 0: administration of Pregnant mare's serum gonadotropin (PMSG); day 1: administration of hCG and mating with stud male; day 3: female culled 12 hours post-coitum and ovaries collected. The external capsula was removed, the ovaries were dissected and the cells were dissociated by treating them for 10 minutes in collagenase + dispase, then 2 X 10 minutes in trypsin. The OSE cells were then cultured in 25% DMEM, 25% F12, 25% MCDB-105, 25% 199 medium in the absence of antibiotics.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cells at 80% confluence were collected by cell scraping in ice cold PBS, spun down for 5 minutes at 1000 rpm at 4deg, snap frozen and stored at -80deg. Total RNA was extracted using the Macherey-Nagel RNA L Kit, following the manufacturer's recommendations, which includes a DNAseI treatment step. Elution was made in RNAse-free wtaer. The RNA quality was checked using the Agilent 2100 bioanalyzer. The RNA concentration was measured using the Vysis Nanodrop.
|
| Label |
Cy5,Cy3
|
| Label protocol |
The amplification of the extracted RNA was made using the Amino Allyl MessageAmp aRNA kit (Ambion, 1752), following the manufactirer's instructions, starting from 4ug of total RNA per sample. The labelling was made using Cy3 Mono-Reactive Dye (Amersham, PA23001) and Cy5 Mono-Reactive Dye (Amersham, PA25001). RNA and Dye concentrations for each sample were determined using the Vysis Nanodrop.
|
| |
|
| |
| Hybridization protocol |
The Hybridisation kit (Agilent, 5184-3468) was used, following the manufacturer's instructions. The hybridisation was conducted with the dye-swap technique, and for each slide were used 0.5mg of Cy3- or Cy5-labelled cRNA from each of the two samples.
|
| Scan protocol |
The Agilent G2565A Microarray Scanner with the Agilent G2567AA Feature Extraction Software were used.
|
| Description |
Analysis used epithelial OSE amplified RNA (control) for comparison to the mesenchymal (experimental) taken after EMT induced by changing the culture conditions from 2.5% FBS to 15% FBS for one week.
|
| Data processing |
The data were background corrected using the method called minimum in the Limma R library for microarray data analysis within R. In order to normalise all arrays in the same manner, we used a global LOWESS fit to normalise each array. The linear model functionality in Limma was used to combine the replicate array data for the comparison (epithelial versus mesenchymal): the linear model output gives an overall estimate for the differential expression between the two samples for each gene.
|
| |
|
| Submission date |
Jan 19, 2010 |
| Last update date |
Jan 19, 2010 |
| Contact name |
Cristian Brocchieri |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Cambridge
|
| Street address |
Hills Road
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 0QH |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL922 |
| Series (1) |
| GSE19947 |
Transcriptional profile of Epithelial to Mesenchymal Transition in the Ovarian Surface Epithelium |
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