|
| Status |
Public on Jan 01, 2015 |
| Title |
HeLa_heat-shock_vs_control_rep1_dye-swap |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
heat-shock_control_rep1_dye-swap
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa cell
treatment: control RNA
|
| Treatment protocol |
Heat shock stress was induced by subjecting the cells to the temperature of 45°C for 30 minutes, in water bath. Cells were subsequently transferred to the incubator for two hours. Before subjecting to heat shock, cells were washed with 1X PBS twice and then fresh media was added for proper quantization of heat shock response.
|
| Growth protocol |
HeLa cell line was used which was procured from National Center for Cell Science, Pune, India. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with heat inactivated 10% fetal bovine serum (FBS) from GIBCO and 1% antibiotic antimycotic solution from HiMedia, at 37°C in 5% CO2 and 95% atmosphere incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA used for the whole-genome expression profiling of miRNA was isolated using Trizol (Invitrogen), according to the manufacturer’s instructions. Further, RNA was purified using RNeasy purification kit from Qiagen to remove residual aromatic compounds that may hinder with microarray expression.
|
| Label |
Cy5
|
| Label protocol |
1µg of total RNA was used for 5'-dephosphorylation of miRNAs using calf intestinal phosphatase (CIP). Spike-in miRNAs were also added for experimental control. Subsequently, samples were fluorescently labeled with Hy3 or Hy5 to the 3'-end of the miRNAs. The complementary samples labeled with Hy3 or Hy5 were combined on ice and added onto the slide placed in the slide chamber for hybridization.
|
| |
|
| Channel 2 |
| Source name |
heat-shock_treated_rep1_dye-swap
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
treatment: heat-shock treated RNA
|
| Treatment protocol |
Heat shock stress was induced by subjecting the cells to the temperature of 45°C for 30 minutes, in water bath. Cells were subsequently transferred to the incubator for two hours. Before subjecting to heat shock, cells were washed with 1X PBS twice and then fresh media was added for proper quantization of heat shock response.
|
| Growth protocol |
HeLa cell line was used which was procured from National Center for Cell Science, Pune, India. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with heat inactivated 10% fetal bovine serum (FBS) from GIBCO and 1% antibiotic antimycotic solution from HiMedia, at 37°C in 5% CO2 and 95% atmosphere incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA used for the whole-genome expression profiling of miRNA was isolated using Trizol (Invitrogen), according to the manufacturer’s instructions. Further, RNA was purified using RNeasy purification kit from Qiagen to remove residual aromatic compounds that may hinder with microarray expression.
|
| Label |
Cy3
|
| Label protocol |
1µg of total RNA was used for 5'-dephosphorylation of miRNAs using calf intestinal phosphatase (CIP). Spike-in miRNAs were also added for experimental control. Subsequently, samples were fluorescently labeled with Hy3 or Hy5 to the 3'-end of the miRNAs. The complementary samples labeled with Hy3 or Hy5 were combined on ice and added onto the slide placed in the slide chamber for hybridization.
|
| |
|
| |
| Hybridization protocol |
Samples were incubated for 16-18 hrs. in a water bath. Post-incubation, slides were washed rigorously and scanned.
|
| Scan protocol |
After drying, chips were scanned using Perkin Elmer scanner at 10µM resolution and PMT of 60.
|
| Description |
Dye-swap of replicate 1
|
| Data processing |
The .gpr files were obtained from Perkin Elmer scanner. The arrays were dye-swapped and gene-specific dye-bias correction was applied using the GASSCO method (Margaritis et al., Mol. Sys. Biol. 5:266 (2009)) implemented in the “dyebias” BioConductor package. For normalization, Lowess method is used. The uploaded processed data files contain Lowess normalized log2 test/control ratios.
|
| |
|
| Submission date |
Aug 18, 2014 |
| Last update date |
Jan 01, 2015 |
| Contact name |
Mitali Mukerji |
| E-mail(s) |
mitali@igib.res.in
|
| Phone |
91-11-29879302
|
| Organization name |
CSIR-Institute Of Genomics and Integrative Biology
|
| Department |
Functional Genomics and Molecular Medicine
|
| Lab |
319
|
| Street address |
Sukhdev Vihar, Mathura Road
|
| City |
New Delhi |
| State/province |
New Delhi |
| ZIP/Postal code |
110020 |
| Country |
India |
| |
|
| Platform ID |
GPL9908 |
| Series (1) |
| GSE60472 |
Whole genome miRNA expression profiling using HeLa cells in response to Heat shock stress. |
|
