cell line: Hela cells, CCL-2 agent: none time point: 0 min population: subpolysomal RNA biological replicate: #3
Biomaterial provider
ATCC
Treatment protocol
HeLa cells were seeded on adherent plates and serum starved for 12h with DMEM, 0.5% FBS, 2mM glutamine.
Growth protocol
HeLa cells were cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2mM glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin at 37 °C in a humidified atmosphere of 5% CO2.
Extracted molecule
total RNA
Extraction protocol
Cells were washed once with phosphate buffer saline (PBS) and treated directly on the plate with 300 µl lysis buffer [10 mM NaCl, 10 mM MgCl2, 10 mM Tris–HCl, pH 7.5, 1% Triton X-100, 1% sodium deoxycholate, 0.2 U/µl RNase inhibitor (Fermentas) and 1 mM dithiothreitol] and transferred to an Eppendorf tube. After a few minute incubation on ice with occasional vortexing, the extracts were centrifuged for 10 min at 12000 g at 4°C. The supernatant was stored at –80°C or loaded directly onto a 15–50% linear sucrose gradient containing 30 mM Tris–HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, and centrifuged in an Sorvall rotor for 120 min at 40000 rpm. Subpolysomal fractions were collected monitoring the absorbance at 254 nm and treated directly with proteinase K. After phenol–chloroform extraction and isopropanol precipitation, subpolysomal RNA was resuspended in 30 µl of water and then repurified with RNeasy kit (Qiagen, Hilden, Germany). RNA quality was assessed by agarose gel electrophoresis and the Agilent 2100 Bioanalyzer platform.
Hybridized microarray slides were scanned with an Agilent DNA Microarray Scanner (G2505C, Agilent Technologies, Santa Clara, CA) at 5 micron resolution with the manufacturer’s software (Agilent ScanControl 8.1.3). The scanned TIFF images were analyzed numerically using the Agilent Feature Extraction Software version 10.7.7.1 according to the Agilent standard protocol GE1_107_Sep09.
Description
Subpolysomal RNA sample extracted from serum starved HeLa cells.
Data processing
Agilent Feature Extraction Software version 10.7.7.1 (standard protocol GE1_107_Sep09) was used for data extraction, background correction and flagging of non-uniform features. The output of Feature Extraction was analyzed with the R software environment for statistical computing (http://www.r-project.org/) and the Bioconductor library of biostatistical packages (http://www.bioconductor.org/). Signal intensities across arrays were normalized with the quantile normalization algorithm.
