Title: Affymetrix CEL analysis. Description:
Affymetrix Generic Hybridization
nucleic acid labeling protocol
Labeling of samples was carried out according to the manufacturer’s standard protocol
The FRT cells and all derivatives were cultured in Coon's modified Ham's F12 medium with the addition, when appropriate, of antibiotic used to select for integration of DNA expression vectors.
(Parameters: time unit = seconds, temperature unit = C)
Transfections were performed by using the FuGENE 6 Transfection Reagent (Roche). Subconfluent cells (ca. 3x106), cultured in 100 mm dishes, were treated for 24 h with 10 ml of medium containing transfection solution. The transfection solution was prepared by incubating the Dulbecco's Modified Eagle Medium (DMEM), FuGENE 6 Transfection Reagent and DNA (3-4 ug) in a volumetric proportion of 20:3:1, for 40 min at RT. Then the cells were split in the ratios of 1:10 and 1:5 into 100 mm dishes, adding to the culture medium, the specific antibiotic such as: 600 ug/ml G418 (Neomycin) and 1 ug/ml puromycin dihydrochloride. Half these amounts were used thereafter to maintain the cultures. After about 14 days of culture in the presence of the appropriate selection medium, individual clones were isolated and examined by Western Blot analysis and Immunofluorescence.
Total RNA was extracted from about 6x10^6 cells, plated in 100 mm diameter plastic dishes, using TRIzol Reagent (Invitrogen). Cells were washed three times with PBS, treated with ice-cold TRIzol Reagent for 5 min, then scraped and the cell lysate was collected in tube, to which 0.2 volume of chloroform was added. After extraction and centrifugation, the aqueous layer was transferred to another tube and precipitated with 500 ul of isopropanol at -20ﾰC for at least 30 min. After a 70% ethanol wash and air-drying, the pellet was resuspended in RNAse-free H2O. Finally, the samples were treated with DNAse in order to eliminate residual genomic DNA. The total RNA then was purified by the RNAeasy mini kit (Qiagen) and quantified using Nano Drop instrument.
(Parameters: Extracted product = total_RNA, Amplification = none)