|Public on Mar 31, 2010
|p53shRNA 10A_SH (A)
|SV40 ts-LTA-hTERT oral keratinocytes, p53 stably downregulated by p53shRNA
|Cells were cultured at 39 °C for three weeks prior RNA extraction.
|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)
|Total RNA was isolated with Trizol (Invitrogen, breda, The Netherlands), according to the manufacturer's instructions. Total RNA was further analyzed on the bioanalyzer (Agilent) and only RIN values of 8 or above were included.
|500 ng of total RNA was reverse transcripted to cDNA using T7 Promoter Primer and MMLV-RT. Then the cDNA was converted to aRNA using T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3- or cyanine 5-labeled CTP (Two-Color Microarray-Based Gene Expression Analysis Protocol, Agilent).
|825ng of Cy3 and Cy5 labelled aRNA were hybridized competitively for 17 hrs in a 65 °C 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).
|Arrays were scanned at 5um resolution on an Agilent DNA Microarray Scanner (G2505B, Agilent) using the default settings for 4x44k format two-color arrays.
|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
|Aug 27, 2008
|Last update date
|Mar 31, 2010
|+31 20 4442428
|Vrije Universiteit Medical Center
|Microarray Core Facility
|De Boelelaan 1117
|Gene expression profiles following TP53 downregulation