|
| Status |
Public on Apr 01, 2012 |
| Title |
HCT116_control_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
HCT116, control, replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116 cells
|
| Treatment protocol |
2 x 105 of HCT116 cells were transfected with 4 pmol of control oligonucleotide or mir-493 mimic for 24 hrs.
|
| Growth protocol |
HCT116 cells were grown in DMEM medium supplemented with 10% heat-inactivated fetal calf serum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs from the transfected HCT116 cells were extracted using miRNeasy mini kit (QIAGEN) according to the manufacturer’s protocol. RNA was quantified using a NanoDrop-1000 spectrophotometer. RNA quality control was performed in 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA).
|
| Label |
Cy3
|
| Label protocol |
Cy3-labeled cRNA was prepared from 0.2 ug RNA using Quick-Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy mini kit (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after control transfection
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1(Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 026652_D_F_20100430) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Aug 30, 2011 |
| Last update date |
Apr 01, 2012 |
| Contact name |
koji okamoto |
| E-mail(s) |
kojokamo@ncc.go.jp
|
| Phone |
81335422511
|
| Organization name |
National Cancer Center Research Institute
|
| Department |
Division of Cancer Differentiation
|
| Street address |
5-1-1 Tsukiji
|
| City |
Chuo-ku |
| State/province |
Tokyo |
| ZIP/Postal code |
1040045 |
| Country |
Japan |
| |
|
| Platform ID |
GPL13497 |
| Series (1) |
| GSE31751 |
Regulation of gene expression by mir-493 |
|
