|
| Status |
Public on May 09, 2014 |
| Title |
HT-29_3D_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
HT-29_3D culture Matrigel on top_7 days
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HT-29
cell type: colorectal cancer cells
culture condition: 3D culture Matrigel on top_7 days
|
| Growth protocol |
For 3D-culture, cells were plated in the Matrigel (BioCoat Matrigel Basement Membrane, growth factor reduced, BD Biosciences, Heidelberg, Germany) on-top assay at a density of 18000 cells per well in 24 well plates. Spheres were cultured for 7 days before recovering from Matrigel. Medium was changed every other day in 3D cultures. For 2D-culture, cells were maintained under standard tissue-culture conditions in RPMI 1640 + GlutaMAX-I (Gibco/Invitrogen, Darmstadt, Germany) containing 10 % fetal calf serum (Gibco/ Invitrogen) and were harvested after seven days at a confluency of around 80%.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
3D-spheres were recovered from the Matrigel Basement Membrane by removing the medium from the Matrigel cell culture and incubation in 400 µl/well dispase (BD Biosciences, Heidelberg, Germany), preheated at 37°C, and incubated for 2 h at 37°C and 5% CO2. The activation of the dispase was stopped with 10 mM EDTA in 1xPBS. Spheres were centrifuged and washed with 1xPBS. 2D cultured cells were also washed with PBS and scraped off the culture dish. Total RNA was isolated using RNeasy Mini Kit (Qiagen) according to manufacturer's instructions. RNA was diluted in 50-80 µl nuclease free and sterile water. RNA concentration was measured spectrophotometrically at 260 nm and RNA quality was assessed using the Agilent 2100 Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Synthesis of cDNA and subsequent fluorescent labeling of cRNA was performed according to the manufacturers´ protocol (One-Color Microarray-Based Gene Expression Analysis, Low Input Quick Amp Labeling, Vers. 6.5; Agilent Technologies). Briefly, 100 ng of total RNA were converted to cDNA, followed by in vitro transcription and incorporation of Cy3 labeled nucleotides into newly synthesized cRNA
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| |
|
| Hybridization protocol |
After fragmentation, labeled cRNA was hybridized to Agilent Human 8x60K High Density Oligonucleotide Microarrays for 17h at 65°C according to the manufacturers´ protocol (One-Color Microarray-Based Gene Expression Analysis, Low Input Quick Amp Labeling, Vers. 6.5; Agilent Technologies)
|
| Scan protocol |
Microarray slides were scanned as described in the manufacturers´ protocol (Scanner G2505C, Agilent Technologies).
|
| Description |
9HT29_3D_US45103065_252800411145_S01_GE1_107_Sep09_1_2
|
| Data processing |
Signal intensities on 20 bit tiff images were calculated by Feature Extraction software (FE, Vers. 10.7.1.1; Agilent Technologies) using scan protocol GE1_107_Sep09 / grid 028004_D_F_20100430. Data analyses were conducted with GeneSpring GX (Vers. 10.5; Agilent Technologies). Probe signal intensities were quantile normalized across all samples to reduce inter-array variability. Input data pre-processing was concluded by baseline transformation to the median of all samples.
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| |
|
| Submission date |
May 08, 2014 |
| Last update date |
May 09, 2014 |
| Contact name |
Karl Koehrer |
| E-mail(s) |
rene.deenen@hhu.de
|
| Organization name |
University of Duesseldorf
|
| Department |
BMFZ
|
| Lab |
GTL
|
| Street address |
Universitaetsstr. 1
|
| City |
Duesseldorf |
| ZIP/Postal code |
40225 |
| Country |
Germany |
| |
|
| Platform ID |
GPL14550 |
| Series (1) |
| GSE57446 |
Impact of the 3D Microenvironment of Phenotype, Gene Expression, and EGFR Inhibition of Colorectal Cancer Cell Lines |
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