|
| Status |
Public on Dec 06, 2010 |
| Title |
EP156T cells cultured 5 days in GFR Matrigel. Rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
GFR Matrigel 3D culture
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: EP156T
organ: Prostate
cell line origin: Non-transformed prostate epithelial cells immortalized with pBabe-hTERT-puro retroviral vector
days in 3d: 5
|
| Treatment protocol |
Monolayer cultures were propagated in 10 cm cell culture dishes. 3D prostaspheres were cultured in Millicell hanging cell culture inserts with 1.0 µm PET transparent membranes (Millipore) on 6-well plates (Costar). The membranes were pre-coated with matrigel/medium mix (1:1) and incubated at 37˚C for 1 hour, to prevent cells from growing as a monolayer on the membrane. Cell suspension was mixed 1:4 with matrigel and transferred onto the coated membrane, polymerized overnight at 37˚C. Fresh medium was added every other day.
|
| Growth protocol |
All cells were propagated at 37˚C in standard cell culture conditions (5% CO2, 95% humidity). Non-transformed cells and their derivatives (EP156T, RWPE-1, PrEC and RWPE-2/w99) were cultured in Keratinocyte Serum-Free Medium (KSFM) supplemented with 2% (v/v) fetal bovine serum, 12.5 mg/l bovine pituitary extract (BPE) and 1.25 µg/l EGF. All transformed cells (PC-3, PC-3M, ALVA31, DU145, LNCaP, 22rv1)were cultured in RPMI-1640 supplemented with 10% (v/v) fetal bovine serum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
3D prostasphere cultures were washed with ice-cold PBS, membranes excised with a scalpel, and gels (plus prostaspheres) transferred into 6-well plates. The gels were mixed vigorously with 5 mM EDTA in PBS, transferred into Falcon tubes, and incubated on a tabletop rocker for 45 minutes, to detach prostaspheres from Matrigel. Free prostaspheres were sedimented by centrifugation and lysed with RLT buffer (Qiagen). Cells propagated in monolayer were lysed at 90% confluence, directly on 10cm cell culture dishes, with RLT buffer. RNA was extracted with QIAGEN RNeasy Mini kit in accordance with the prescribed protocol provided with the kit. Quality control was performed with Agilent Bioanalyser.
|
| Label |
biotin
|
| Label protocol |
Total RNA was amplified with Ambion's Illumina TotalPrep RNA Amplification kit (Ambion). In vitro transcription reaction was performed overnight to yield sufficiently biotinylated cRNA. The cRNA concentrations where checked with Nanodrop ND-1000 and cRNA quality was controlled by BioRad’s Experion electrophoresis station.
|
| |
|
| Hybridization protocol |
Samples were hybridized to Illumina’s Sentrix HumanRef-8 v3 Expression BeadChips, at 58 °C overnight according to Illumina Whole-Genome Gene Expression Direct Hybridization Guide.
|
| Scan protocol |
Chips were scanned with Illumina BeadArray Reader.
|
| Description |
replicate 2
|
| Data processing |
Raw microarray data were quantile-normalized and log2 transformed using the Beadarray R package
|
| |
|
| Submission date |
Dec 11, 2009 |
| Last update date |
Dec 11, 2009 |
| Contact name |
Matthias Nees |
| E-mail(s) |
matthias.nees@vtt.fi
|
| Phone |
+358 40 8314 839
|
| Fax |
+358 2 720 2840
|
| URL |
http://www.vtt.fi
|
| Organization name |
VTT Technical Research Centre of Finland
|
| Department |
Medical Biotechnology
|
| Street address |
Itäinen Pitkäkatu 4C
|
| City |
Turku |
| ZIP/Postal code |
20521 |
| Country |
Finland |
| |
|
| Platform ID |
GPL6883 |
| Series (1) |
| GSE19426 |
Comprehensive Characterization of 3D Models for Prostate Cancer Growth and Invasion in Laminin-rich Extracellular Matrix |
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