|
| Status |
Public on Mar 31, 2010 |
| Title |
p53-dominant negative mutation (arg175His) 19A_DN (B) |
| Sample type |
RNA |
| |
|
| Source name |
19A_DN
|
| Organism |
Homo sapiens |
| Characteristics |
SV40 ts-LTA-hTERT oral keratinocytes, p53 stably downregulated by p53-dominant negative mutation (arg175His)
|
| Treatment protocol |
Cells were cultured at 39 C for three weeks prior to RNA extraction.
|
| Growth protocol |
SV40 ts-LTA-hTERT oral keratinocytes were maintained in Keratinocyte-SFM supplemented with bovine pituitary extract (25 mg/ 500 ml) human recombinant EGF (2.5 ug/500 ml medium)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with Trizol (Invitrogen Cat. No. 15596-026, Carlsbad, CA), according to the manufacturer's instructions. Total RNA was further purified using the Qiagen RNAeasy Mini Kit (Cat. No. 74104) according to the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
500ng of RNA was linearly amplified and fluorescently labeled with either Cy3-CTP or Cy5-CTP with the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Amstelveen, The Netherlands).
|
| |
|
| Hybridization protocol |
825ng of Cy3 and Cy5 labelled aRNA were hybridized competitively for 17 hrs in a 65C hybridization oven (G2545A, Agilent) set to 10 rpm in a final concentration of 1X GEx Hybridization Buffer HI-RPM, according to the manufacturer's recommnded protocol (Two-Color Microarray-Based Gene Expression Analysis, Agilent). Arrays were washed according to the manufacturer's recommended protocol (Two-Color Microarray-Based Gene Expression Analysis, Agilent).
|
| Scan protocol |
Arrays were scanned at 5um resolution on an Agilent DNA Microarray Scanner (G2505B, Agilent) using the default settings for 4x44k format two-color arrays.
|
| Description |
na
|
| Data processing |
Images were auto gridded, analyzed and data extracted using Agilent Feature Extraction software (version 8.5.1). Raw expression data generated by the Feature Extraction software was imported into R using the LIMMA package in Bioconductor. The intensity distributions within and between arrays were normalized using the quantile scaling algorithm in LIMMA. After normalization, the separate intensity channels were extracted from the ratio measurements
|
| |
|
| Submission date |
Aug 27, 2008 |
| Last update date |
Nov 16, 2011 |
| Contact name |
Daoud Sie |
| E-mail(s) |
d.sie@vumc.nl
|
| Phone |
+31 20 4442428
|
| Organization name |
Vrije Universiteit Medical Center
|
| Department |
Pathology
|
| Lab |
Microarray Core Facility
|
| Street address |
De Boelelaan 1117
|
| City |
Amsterdam |
| ZIP/Postal code |
1081 HV |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE12553 |
Gene expression profiles following TP53 downregulation |
|
