|
| Status |
Public on Apr 01, 2010 |
| Title |
Control |
| Sample type |
RNA |
| |
|
| Source name |
untreated Y79 cells (control)
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Y79 retinoblastoma cells
|
| Treatment protocol |
Y79 cells were treated with 20uM curcumin for 48hrs.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using using mirVana miRNA Isolation kit (Ambion, AM1560) following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
miRNA labeling was performed as described in Agilent miRNA microarray protocol version 2.0 using Agilent miRNA labeling kit. 100 ng of total RNA was dephosphorylated with calf intestine alkaline phosphatase for 30 min at 37°C. Denaturation was performed by adding DMSO and incubating at 100°C for 7 min and immediately transferred to ice water bath. Ligation was performed with pCp-Cy3 at 16 °C for 2 h. The labeled samples were dried completely in a vaccum concentrator and resuspended in 18 ul of nuclease free water.
|
| |
|
| Hybridization protocol |
The hybridization mixture [10X GE blocking agent (4.5 ul), 2X Hi-RPM hybridization buffer (22.5ul)] along with labeled miRNA sample was heated for 5 min at 100 °C and immediately cooled to 0 °C. Each 45 mL sample was hybridized onto a microarray at 55 °C for 20 h. Slides were washed 5 min in GE wash buffer 1 at RT and again for 5 min in GE wash buffer 2 at 37 °C, followed by an acetonitrile wash for 1 min at RT to dry the slides completely.
|
| Scan protocol |
Scanned on an Agilent G2505C scanner and Images were quantified using Agilent Feature Extraction Software (version 10.5.1.1)
|
| Description |
untreated Y79 cells (control)
|
| Data processing |
The normalization was done using GeneSpring GX 11 Software. Recommended Percentile Shift Normalization and samples normalized with respect to control samples. Significant miRNA’S up and down regulated showing one fold and above among the groups was identified. Differentially regulated miRNA were clustered using hierarchical clustering to identify significant expression patterns. Using GeneSpring software miRNA targets was identified.
|
| |
|
| Submission date |
Mar 30, 2010 |
| Last update date |
Mar 30, 2010 |
| Contact name |
Genotypic technology |
| E-mail(s) |
sudha.rao@genotypic.co.in
|
| Organization name |
Genotypic Technology
|
| Street address |
259, Apoorva 4th cross,80 feet Road,RMV 2ND STAGE
|
| City |
Bangalore |
| State/province |
Karnataka |
| ZIP/Postal code |
560094 |
| Country |
India |
| |
|
| Platform ID |
GPL7731 |
| Series (1) |
| GSE21126 |
Y79 retinoblastoma cells: control vs. curcumin treated |
|
