agent: curcumin age: 1 to 3 day aged adult strain: Canton-S gender: 30 male and 30 female
Treatment protocol
Stock solutions of curcumin from Agros (Geel, Belgium) were prepared and added to the fly food and all feeding assays were performed in 500 mL cages under 12 hour light/dark cycle. Assays were performed in 50% humidity and 25 °C. Adult sucrose-yeast diets were prepared with 10 g sucrose, 10 g yeast, 1.1 mL of 20% tegosept (w:v in ethanol), and 0.79 g agar per 100 ml in water. After boiling, media was cooled to 60 °C and curcumin stock solution, or ethanol alone, was added by volume of 19:1. Then 5 ml of media was distributed into 10 X 75 mm shell vials, stored at 4 °C and used within two to three weeks.
Growth protocol
Larvae of the Canton-S strain were grown on standard CSY medium [5.2 g cornmeal, 11 g sucrose, 11 g nutritional yeast (MP Biomedicals, Solon, OH), 1.1 mL of 20% tegosept, 0.79 g agar per 100 ml in water] supplemented with several grains of live yeast.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from each sample using Trizol (Invitrogen) according to the manufacturer’s protocol. For clean-up the RNA samples, the RNA was subjected to purify using the RNeasy Maxi Kit (Qiagen, Valencia, CA) according to the manufacturer's directions. The quality and yield of the preparation was assessed throughout processing and labeling using a 2100 Bioanalyzer (Agilent, Santa Clara, CA).
Label
biotin
Label protocol
Synthesis of cDNA and biotin-labeled cRNA, fragmentation, hybridization, washing, and scanning were performed according to the Affymetrix GeneChip Expression Analysis Technical Manual (2005). Double-stranded cDNA was synthesized from 5 ug of template total RNA using One-Cycle cDNA Synthesis kit (Affymetrix, U.S.A.), and biotin labeled cRNA was synthesized using IVT Labeling kit (Affymetrix, U.S.A.). The labeled cRNA was purified with GeneChip® Sample Cleanup Module (Affymetrix, U.S.A.). The quality and quantity of the cRNA was checked by conducting gel electrophoresis and NanoDrop® ND-1000 (NanoDrop, U.S.A.), respectively.
Hybridization protocol
Fifteen micrograms of the purified cRNA of each sample was fragmented and hybridized to arrays for 16 h at 45℃. Biotinylated cRNA probes were hybridized to the high density Drosophila GeneChip 2.0 oligonucleotide microarrays (Affymetrix, Inc., Santa Clara, CA, USA) and visualized with a streptavidin-phycoerythrin conjugate.
Scan protocol
All arrays were washed and stained automatically by using a fluidics Station 450 (Affymetrix, U.S.A.) and scanned by GeneChip® scanner 3000 (Affymetrix, Inc, U.S.A.).
Description
Gene expression data from aged and curcumin feeding.
Data processing
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method.
