tissue: human skin fibroblasts gender: male young/old: young batch: 3
Treatment protocol
To chronically stress fibroblast strains, medium was supplemented for 3 hours and 3 days with 0.6 μM rotenone (Sigma, St Louis, MO, USA), known to induce an increase in the intracellular production of ROS at the mitochondrial level
Growth protocol
Three-mm (Leiden 85-plus Study) were taken from the sun unexposed medial side of the upper arm. Fibroblasts were grown in D-MEM:F-12 (1:1) medium supplemented with 10% fetal calf serum (FCS), 1 mM MEM sodium pyruvate, 10 mM HEPES, 2 mM glutamax I, and antibiotics (100 Units/mL penicillin, 100 μg/mL streptomycin, and 0.25–2.5 μg/mL amphotericin B), all obtained from Gibco, Breda, the Netherlands. Different FCS batches were used for fibroblasts from the Leiden 85-plus Study (Gibco, batch no. 40G4932F) and for fibroblasts from the LLS (Bodinco, Alkmaar, the Netherlands, batch no. 162229). This medium will be referred to as standard medium. Fibroblasts were incubated at 37oC with 5% CO2 and 100% humidity. All cultures that are used in the present study were grown under predefined, highly standardized conditions as published earlier {Maier, 2007 118 /id} and frozen at low passage. Trypsin (Sigma, St Louis, MO, USA) was used to split fibroblasts using a 1:4 ratio each time they reached 80-100% confluence.
Extracted molecule
total RNA
Extraction protocol
Total mRNA was isolated using the RNeasy Mini Kit (Qiagen Ltd, Crawley, UK)
Label
Cy3
Label protocol
300ng mRNA was mixed with an appropriate amount of One-Color RNA Spike-In RNA and converted into labelled cRNA (One-Color Low RNA Input Linear Amplification Kit PLUS).
Hybridization protocol
Labelled cRNA was purified using an RNeasy Mini Kit (Qiagen Ltd, Crawley, UK) and 2μg was hybridised to Agilent human whole genome Oligo Arrays (G4112F) using reagents supplied in the Agilent Hybridisation Kit (One-Color Microarray-Based Gene Expression Analysis Protocol). Microarray slides were hybridised for 17 h at 65 ºC and subsequently washed in acetonitrile for 1 min followed by 30s in Agilent Stabilisation and Drying Solution.
Scan protocol
Scanning of the slides was performed with the Agilent G2565BA Microarray Scanner System. The Agilent G2567AA Feature Extraction Software (v.9.1) was used to extract data and check the quality.
Description
Gene expression after 3 hours rotenone treatment
Data processing
The Agilent G2567AA Feature Extraction Software (v.9.1) was used to extract data and check the quality. The data was background corrected by Normexp+offset, quantile normalised and log transformed: # normalise # background correction - normexp + offset method dat2<- backgroundCorrect(ddaux,"normexp",offset=50) # quantile dat2<-normalizeBetweenArrays(dat2$R,method="quantile") # ln dat2<-log(dat2)
