|
| Status |
Public on Sep 26, 2015 |
| Title |
PHBECs_MP9_coculture_Array4_replicate1 |
| Sample type |
RNA |
| |
|
| Source name |
primary human breast epithelial cells, dissociated co-culture
|
| Organism |
Homo sapiens |
| Characteristics |
reduction mammoplasty: MP9
cell type: co-culture derived epithelial cells
|
| Treatment protocol |
Serum free medium (M5) was supplemented and/or replaced two to three times a week during the whole ex-vivo culture period. After 30 days the cultures were dissociated with mild proteinase/EDTA (HyQtase, Thermo Scientific) treatment for 20 min. Cells remained thereafter on ice and FACS sorts were performed immediately to isolate viable epithelial or stromal cells, based on their fluorescent protein tag. 300 cells of each condition were directly sorted in 100ul TRIZOL in cooled tubes. The samples were immediately frozen on dry ice and stored at -70°C prior to RNA extraction.
|
| Growth protocol |
Human primary epithelial progenitor derived cells and mesenchymal stem cells derived cells bearing fluorescent tags were either co-cultured in novel ex vivo culture conditions on ECM coated meshes in serum free medium (M5) or cultured as monocultures in the same conditions for 30 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from 300 sorted cells with the PicoPure RNA isolation kit (Arcturus)
|
| Label |
biotin
|
| Label protocol |
Reverse transcription was performed using 4 μM T7- (dT)24/T7- (dN)6 primer mix (Affymetrix) and 150 units of Superscript II reverse transcriptase (Invitrogen). The synthesis of second-strand cDNA was performed by mixing 4 mM dNTPs, 6 units DNA polymerase I, and 0.4 units RNase H in a 20-µL reaction volume. cRNA was produced by in vitro transcription with a T7 RNA polymerase at 37°C for 14 h using the MEGAscript T7 kit (Ambion) as per the manufacturer's instructions. For the second cycle, the first-strand cDNA was synthesized using 0.2 μg random primers from 9 μL of purified cRNA. The second-strand cDNA was produced using 10 µM T7- (dN)6 primer and 40 units DNA polymerase at 16°C for 2 h, after which 10 units T4 DNA polymerase (Invitrogen) were added and the incubation continued for another 10 min. The cDNA was in vitro transcribed with T7 RNA polymerase at 37°C for 16 h. The single-strand cDNA was synthesized using 10 µg purified cRNA in the presence of 4 µg random primers, 0.2 M DTT, 12 mM dNTP + dUTP and 750 units Superscript II (Roche Diagnostics) in a total volume of 20 µL. The cRNA was hydrolyzed with 2 units RNase H at 37°C for 40 min. The sense cDNA was purified and eluted in 28 µL elution buffer. Amplified products were purified using the GeneChip cDNA Sample Cleanup Module (Affymetrix) with a 6,000 g centrifugation during the first two steps. To improve recovery from the columns, water or elution buffer were spun into the matrix at 50 g and left to stand for 4 min before a 16,000 g centrifugation. The quantity and purity of the cRNA and cDNA produced during the first and second rounds were evaluated using a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies). The cDNA was then fragmented by uracil DNA glycosylase and apurinic/apyrimidic endonuclease 1 and biotin-labeled with terminal deoxynucleotidyl transferase using the GeneChip WT Terminal labeling kit (Affymetrix).
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| |
|
| Hybridization protocol |
Hybridization was carried out with 5 μg of biotinylated target, which was incubated with a GeneChip human Gene 1.0 ST Array (Affymetrix) at 45°C for 16 h. Following hybridization, non-specifically bound nucleotides were removed by washing and specifically bound target detected using a GeneChip Hybridization, Wash and Stain kit and a GeneChip Fluidics Station 450 (Affymetrix).
|
| Scan protocol |
The arrays were scanned using a GeneChip Scanner 3000 7G (Affymetrix) and CEL files acquired using GeneChip Command Console Software (Affymetrix).
|
| Data processing |
Arrays were normalized and probeset-level expression values calculated with R/Bioconductor's (v2.14) 'affy' package using the rma() function (www.bioconductor.org).
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| |
|
| Submission date |
Sep 26, 2013 |
| Last update date |
Sep 26, 2015 |
| Contact name |
Tim Roloff |
| E-mail(s) |
tim.roloff@fmi.ch
|
| Organization name |
FMI
|
| Department |
Functional Genomics
|
| Street address |
Maulbeerstrasse 66
|
| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE51201 |
Gene expression profiles of epithelial and mesenchymal cells extracted from heterotypic ex vivo co-cultures or monocultures |
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