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Status |
Public on Jun 01, 2007 |
Title |
Quercetin day 05 sample 1 |
Sample type |
RNA |
|
|
Source name |
Human colon cancer cell line Caco-2
|
Organism |
Homo sapiens |
Characteristics |
The Caco-2 cell line is derived from a male Caucasian with a colon tumor
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Biomaterial provider |
American Type Culture Collection (ATCC)
|
Treatment protocol |
Dulbecco’s Modified Eagle Medium supplemented with 10% (v/v) heat inactivated mycoplasma tested fetal calf serum, 2 mM L-glutamine (6 mM final concentration), 1% (v/v) MEM non-essential amino acids and 50 µg/ml gentamicin. Quercetin in DMEM culture medium was stabilized by 1 mM ascorbate
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Growth protocol |
Caco-2 cells of passage 37 were seeded (1 : 10 split ratio) in triplicate on polycarbonate membrane transwell® inserts (Corning Life Sciences, Cambridge, U.K.), with a membrane diameter of 75 mm (growth area 44 cm2, 0.4 µm pore size) at a density of approx. 40,000 cells/cm2. After 2 days, cell cultures reached confluency, and this time point was considered experimental day 0 and the initial exposure to quercetin. Cells were exposed for a total number of 9 days.
|
Extracted molecule |
total RNA |
Extraction protocol |
On days 5 and 10 post-confluency, cells were first rinsed with ice cold phosphate buffered saline without calcium or magnesium (InvitrogenTM Life Technologies, Breda, The Netherlands). Subsequently, cells were harvested in 2 x 0.75 ml of ice-cold TRIzol (Life Technologies, Paisley, U.K.), immediately frozen in liquid nitrogen and stored at -80°C for up to 2 months, until RNA isolation according to the TRIzol protocol. Following isolation, total RNA was purified with RNeasy Midi columns (QIAGEN, Westburg, Leusden, The Netherlands), including a DNAse incubation step. Quality of purified total RNA was determined on a UV-VIS spectrofotometer (Shimadzu Benelux, ‘s Hertogenbosch, The Netherlands) by calculation of the A260/A280 ratio (1.7 - 2.0) and confirmed on a Lab-on-a-Chip on the Agilent 2100 bioanalyser (Agilent Technologies, Palo Alto, California, U.S.A.) by determining the 28S/18S ribosomal RNA ratio
|
Label |
Biotin
|
Label protocol |
Labeling protocol according to Affymetrix® GeneChip® Expression Analysis Technical Manual
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|
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Hybridization protocol |
Hybridization protocol according to Affymetrix® GeneChip® Expression Analysis Technical Manual
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Scan protocol |
Scan protocol with GeneChip® Scanner 3000 7G, according to Affymetrix® GeneChip® Expression Analysis Technical Manual
|
Description |
Probe sets with an “absent call” (P ≥ 0.065 for HG-U133A 2.0 GeneChip® arrays) across all 8 arrays (n = 7,285) were considered not to be affected by quercetin treatment and/or incubation time and were therefore excluded from further analyses.
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Data processing |
CEL files were imported in Rosetta Resolver 5.0 (Rosetta Inpharmatics LLC, Seattle, U.S.A.), applying data pre-processing (to estimate and reduce systematic errors) and error modeling (for improvement of accuracy of the present and absent calls for low replicate numbers).
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|
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Submission date |
Mar 13, 2007 |
Last update date |
Apr 27, 2007 |
Contact name |
Ashwin Dihal |
E-mail(s) |
[email protected]
|
Phone |
+31 (0)30-6944545
|
Fax |
+31 (0)30 6944989
|
Organization name |
TNO Quality of Life
|
Department |
Business Unit Physiologival Sciences
|
Street address |
Utrechtseweg 48
|
City |
Zeist |
ZIP/Postal code |
3704 HE |
Country |
Netherlands |
|
|
Platform ID |
GPL571 |
Series (1) |
GSE7259 |
Pathway and single gene analysis of Caco-2 cell differentiation by ascorbate-stabilized quercetin |
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