|
| Status |
Public on Apr 18, 2017 |
| Title |
NT Time 24 Hours rep2 |
| Sample type |
RNA |
| |
|
| Source name |
HCT116_NT_24 hrs
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
cell type: Human colon cancer cell line
cell line info.: Originally provided by Dr. B. Vogelstein. Last validation on Dec 2013 by genomic analysis of 18 loci marker (Kit PowerPlex 18D System) verified
treated with: none (untreated control)
time after treatment: 24 hrs
|
| Treatment protocol |
For experiments, cells were seeded at a density of 8×10^3 cells/cm2. The following day, one dish of cells was harvested (NT time-0 condition) and the other cells were treated with DMSO, Peptide-3 or Peptide-3M at a concentration of 10 µM by adding the tretaments directly to the growth medium. Cells were then collected at 24 or 48 hours after tretament. One dish of untreated cells was collected at 24hours.
|
| Growth protocol |
HCT116 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS, Life Technology), 2 mM L-glutamine, and 1% penicillin and streptomycin in a fully-humidified incubator containing 5% CO2 at 37˚C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested after the different treatments and total RNA was isolated from each sample using RNA Purification columns (Norgen, Purification Plus kit).
|
| Label |
cy3
|
| Label protocol |
The cyanine 3-CTP labeled cRNA was prepared using the Agilent Low Input Linear Amplification Kit, following the standard one-color Agilent protocol
|
| |
|
| Hybridization protocol |
One-color mixes of labelled cRNA pairs were hybridized to8x60K whole human genome oligonucleotide microarray (Agilent G4851B, grid ID 039494), at 65°C for 17 hours using Agilent’s Gene Expression Hybridization Kit. The hybridized microarrays were disassembled at room temperature in Agilent Gene Expression Wash Buffer 1. After the disassembly, the microarrays were washed in Gene Expression Buffer 1 for one minute at room temperature, followed by washing with Gene Expression Wash Buffer 2 for one minute at 37°C. The microarrays were then treated with acetonitrile for one minute at room temperature.
|
| Scan protocol |
Post-hybridization image acquisition was accomplished using the Agilent Scanner G2564C. Data extraction from the 20 bits TIFF images was accomplished by Agilent Feature Extraction ver 10.7 software using the standard Agilent one-color gene expression extraction protocol (GE1_107_Sep09).
|
| Description |
SAMPLE 13
|
| Data processing |
The “gProcessedSignal” data column of the Feature Extraction *.txt output file was used, containing the median signals corrected by spatial and multiplicative detrend. Data quality filtering was performed using Microsoft Excel, discarding features with the Feature Extraction flag gIsWellAboveBG=0 in any sample, wich is a statistical method approximately equivalent to discarding features with a Signal/Noise ratio smaller than 2.0-3.0, where Signal = (median of the spot - median spot background level) and Noise is the Standard Deviation of the median spot background. Data were normalized to the 75th percentile in Log2 scale. Then differential gene lists were obtained from filtered data using Microsoft Excel. Differentially expressed genes were identified in Excel by T-test and Log2 foldchange threshold and by functional analysis using Ingenuity Pathway Analysis software.
Data are in Log2 scale filtered and each sample is normalized to the 75th percentile.
|
| |
|
| Submission date |
Oct 30, 2014 |
| Last update date |
Apr 18, 2017 |
| Contact name |
Ivan Arisi |
| E-mail(s) |
i.arisi@ebri.it
|
| Phone |
+39-06-49255230
|
| Organization name |
European Brain Research Institute
|
| Department |
Bioinformatics Facility
|
| Street address |
viale Regina Elena 295
|
| City |
Roma |
| ZIP/Postal code |
00161 |
| Country |
Italy |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE62855 |
Targeting MDM4/MDM2 binding interface for reactivation of p53 in cancer therapy |
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