|
| Status |
Public on Aug 25, 2010 |
| Title |
A498 cells control 75min biological rep3 |
| Sample type |
RNA |
| |
|
| Source name |
A498 cells
|
| Organism |
Homo sapiens |
| Characteristics |
time: 75min
treatment: control
cell line: A498
|
| Treatment protocol |
Cells were treated for 0min, 15min, 60min, 75min, 3hrs, 6hrs and 24 hrs with HAMLET at 0.3mM.
|
| Growth protocol |
A498 cells were cultured in RPMI-1640 with non-essential amino acids (1:100), 1 mM sodium pyruvate, 50 µg/ml Gentamicin (Gibco), 5% fetal calf serum (FCS). Primary cultures of human renal tubular epithelial cells (HRTEC) were cultured in DMEM/F12 with 15% FCS. HAMLET was incubated with cells in serum-free RPMI-1640 at 37°C. FCS was added after 1 hour.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from 3 biological replicates of A498 or HPRTEC cells subjected to control and HAMLET @0.3 treatments using the RNeasy Mini kit (Qiagen) according to manufacturer instructions. RNA quality was assessed on the 2100 Bioanalyser (Agilent) using the RNA 6000 Nano Chip kit (Agilent) for intact 18S and 28S ribosomal peaks without significant degradation (RNA Integrity Number >9) for all samples.
|
| Label |
Biotin
|
| Label protocol |
500ng of total RNA from each sample was reverse transcribed into cDNA and in vitro transcribed into biotin-labelled cRNA using the Illumina TotalPrep RNA Amplification kit (Ambion).
|
| |
|
| Hybridization protocol |
750ng of each cRNA sample was hybridized to HumanWG-6 v3 expression beadchip microarrays (Illumina) as per manufacturer specifications.
|
| Scan protocol |
Microarrays were scanned on the BeadArray Reader (Illumina) at scan factor 1 as per manufacturer specifications.
|
| Description |
Biological replicate 3
|
| Data processing |
The microarray data was subjected to background subtraction on the BeadStudio Data Analysis software (Illumina) and normalized using the Cross-correlation method (Chua et al., 2006). Differentially expressed transcripts were identified based on the mean Log2 fold-change in expression values compared to the wild type ES cell controls with a cutoff at 1.5-fold change.
Background subtracted cross-correlation method normalised intensity values generated using MATLAB scripts
Chua SW, Vijayakumar P, Nissom PM, Yam CY, Wong VV, Yang H: A novel normalization method for effective removal of systematic variation in microarray data. Nucleic acids research 2006, 34(5):e38.
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| |
|
| Submission date |
Aug 24, 2010 |
| Last update date |
Aug 24, 2010 |
| Contact name |
Henry Yang |
| E-mail(s) |
henry_yang@immunol.a-star.edu.sg
|
| Organization name |
SIgN
|
| Department |
Bioinformatics Group
|
| Street address |
8A Biomedical Grove
|
| City |
Singapore |
| ZIP/Postal code |
138648 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL6884 |
| Series (1) |
| GSE23772 |
Why does HAMLET preferentially kill tumor cells? p38-dependent death in tumor but up-regulation of innate immunity in healthy, differentiated cells |
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