|
| Status |
Public on Oct 01, 2010 |
| Title |
Control 2h-A |
| Sample type |
RNA |
| |
|
| Source name |
Caco2 cells, 2h, Control
|
| Organism |
Homo sapiens |
| Characteristics |
time point: 2h
agent: none
cell line: Caco-2
|
| Treatment protocol |
Caco-2 cells (4 days post-confluence on transwells) were incubated with Bifidobacterium bifidum PRL2010 (exponentially growing in 8 ml of ‘MRS’ medium (~108 CFU/ml)). 100 µl of bacterial suspension was added to experimental wells while the control wells received the same amount of medium without bacterial cells. Caco-2 cells were harvested for analyses after 2h and 4h of incubation.
|
| Growth protocol |
2x105 Caco-2 cells in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin, streptomycin, amphotericin B and L-glutamine were seeded into the upper compartments of a six-well transwell plate. The lower compartments contained 3.0 ml of the same medium. The cells were incubated at 37 °C in a 5% CO2 atmosphere until they reached three days post-confluence. The cells were then washed with Hank’s solution, and DMEM medium supplemented with L-glutamine, sodium selenite and transferrin was added and incubation continued for a further 24 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with an RNeasy kit (Qiagen) and included a DNAse I step (Qiagen).
|
| Label |
biotin
|
| Label protocol |
Fragmented and biotin-labelled cRNA was synthesized from 8 ug purified RNA using the One-Cycle Target Labeling Kit (Affymetrix) according to the manufacturer's protocol.
|
| |
|
| Hybridization protocol |
Following fragmentation, 15 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Human Genome U133 plus 2.0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station.
|
| Scan protocol |
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner.
|
| Description |
Caco-2 cells (human, Caucasian, colon, adenocarcinoma) were treated with Bifidobacterium bifidum PRL2010 for 2h or 4h, and their gene expression profile was compared to non-stimulated cells.
|
| Data processing |
Initial data analysis was performed using Affymetrix GCOS 1.2 software. Data were further analyzed using Bioconductor (http://www.bioconductor.org) from the R Project for Statistical Computing (http://www.r-project.org). Normalization was performed with gcRMA. The moderated t-test provided by the Bioconductor package limma was used to test for differential expression. Data was considered significant when P<0.05 using the Benjamini and Hochberg false discovery method.
|
| |
|
| Submission date |
May 24, 2010 |
| Last update date |
Aug 12, 2010 |
| Contact name |
Denise Kelly |
| E-mail(s) |
[email protected]
|
| Phone |
+44 1224 716648
|
| Organization name |
University of Aberdeen
|
| Lab |
Gut Immunology Group
|
| Street address |
Institute of Medical Sciences
|
| City |
Aberdeen |
| ZIP/Postal code |
AB25 2ZD |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL7020 |
| Series (1) |
| GSE21976 |
Effects of Bifidobacterium bifidum PRL2010 on gene expression in intestinal epithelial cells |
|
