|
| Status |
Public on Jun 02, 2015 |
| Title |
p53 overexpression_H1299_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
H1299 cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: H1299
treatment: p53 overexpression
tissue: lung
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol-RNA extraction
|
| Label |
Cy3
|
| Label protocol |
miRNA labeling was performed as described in Agilent miRNA microarray protocol version 2.0 using Agilent miRNA labeling kit(Agilent Technologies, Part Number: 5190-0456). 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 hrs. The labeled samples were dried completely in a vacuum 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 incubated for 5 min at 100 °C and immediately kept on ice. Each 45 ul sample was hybridized onto a microarray at 55 °C for 20 h. Slides were washed for 5 min in GE wash buffer 1 at RT and again for 5 min in GE wash buffer 2 at 37 °C. After hybridization, the slides were washed using Gene Expression Wash Buffer1 (Agilent Technologies, Part Number 5188-5325) at room temperature for 5 minutes and Gene Expression Wash Buffer 2 (Agilent Technologies, Part Number 5188-5326) at 37oC for 5 minutes.
|
| Scan protocol |
Scanned on an Agilent G2505C scanner and Images were quantified using Agilent Feature Extraction Software
|
| Description |
miRNAs expression
|
| Data processing |
The normalization was done using GeneSpring GX 12 Software. 90th Percentile Shift Normalization
|
| |
|
| Submission date |
Apr 01, 2014 |
| Last update date |
Jun 02, 2015 |
| 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 |
GPL18509 |
| Series (1) |
| GSE56412 |
Development of micro RNA expression signatures for differential translation of protein isoforms. |
|
