|
| Status |
Public on Dec 20, 2011 |
| Title |
VitD/control 48h rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Control cells 48h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SW480-ADH
treatment: control
time: 48 h
|
| Treatment protocol |
Treatments with 1,25(OH)2D3 or isopropanol (vehicle) were performed in medium supplemented with charcoal-treated serum.
|
| Growth protocol |
A human colon cancer cell line, SW480-ADH, was cultured in DMEM plus 10% foetal bovine serum (Invitrogen, Paisley, UK).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cellular small RNA fractions were extracted with PureLink miRNA isolation kit (Invitrogen) by following manufacturer instructions. Quality of the RNA was determined with a 2100 Bioanalyzer (Agilent Technologies, Carlsbad, CA) and RNA amount was established by using a NanoDrop spectrophotometer (NanoDrop, Wilmington, DE).
|
| Label |
Cy5
|
| Label protocol |
One-step, direct labeling of 500 ng miRNA's extract has been carried out with the Label IT® miRNA Labeling Kit, Cy™3/Cy™5 (Mirus) by following manufacturer instructions. Extracts from cells treated with 1,25(OH)2D3 or vehicle at each time point were compared in dye swapped hybridizations.
|
| |
|
| Channel 2 |
| Source name |
VitD treated cells 48h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SW480-ADH
treatment: vitamin D
time: 48 h
|
| Treatment protocol |
Treatments with 1,25(OH)2D3 or isopropanol (vehicle) were performed in medium supplemented with charcoal-treated serum.
|
| Growth protocol |
A human colon cancer cell line, SW480-ADH, was cultured in DMEM plus 10% foetal bovine serum (Invitrogen, Paisley, UK).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cellular small RNA fractions were extracted with PureLink miRNA isolation kit (Invitrogen) by following manufacturer instructions. Quality of the RNA was determined with a 2100 Bioanalyzer (Agilent Technologies, Carlsbad, CA) and RNA amount was established by using a NanoDrop spectrophotometer (NanoDrop, Wilmington, DE).
|
| Label |
Cy3
|
| Label protocol |
One-step, direct labeling of 500 ng miRNA's extract has been carried out with the Label IT® miRNA Labeling Kit, Cy™3/Cy™5 (Mirus) by following manufacturer instructions. Extracts from cells treated with 1,25(OH)2D3 or vehicle at each time point were compared in dye swapped hybridizations.
|
| |
|
| |
| Hybridization protocol |
Hybridization conditions were as per Mirus' kit recommendations. Slides were pre-soaked (in buffer with 2X SSC, 0.05% SDS, 0.25% Sodium Borohydride) for 30min at 45ºC and pre-hybridized (in buffer with 5X SSC, 0.1% SDS, 1% BSA) at 42°C for 45min. Arrays were hybridized overnight at 42°C simultaneously with Cy5- and Cy3-labeled samples previously denatured in 1X hybridization solution (provided in Mirus' kit). After incubation, slides were washed 2 x 5 min. each in 2X SSC, 0.1% SDS at hybridization temperature, 2 x 5 min. each in 1X SSC at hybridization temperature and finally 2 x 1 min each in 0.1X SSC at room temperature. Slides were briefly dip in dH20 and dried by centrifugation at 500 rcf for 1 minute.
|
| Scan protocol |
The slides were scanned at a resolution of 10 µm with Agilent G2565BA Microarray Scanner (Agilent Technologies) and Cy3 and Cy5 fluorescent signals were quantified using Feature Extraction Software (Agilent Technologies).
|
| Data processing |
Raw data from microarray images were background subtracted and global Lowess normalized with Feature Extraction Software (Agilent Technologies).
|
| |
|
| Submission date |
Dec 19, 2011 |
| Last update date |
Dec 20, 2011 |
| Contact name |
Luis LOMBARDIA |
| E-mail(s) |
llombardia@cnio.es
|
| Organization name |
CNIO (Spanish National Cancer Center)
|
| Department |
Molecualr Pathology Programme
|
| Lab |
Molecular Diagnostics Unit
|
| Street address |
Melchor Fernandez Almagro, 3
|
| City |
MADRID |
| State/province |
Madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
| |
|
| Platform ID |
GPL15042 |
| Series (1) |
| GSE34564 |
MicroRNA-22 upregulation by vitamin D mediates its protective action against colon cancer. |
|
