Agilent 4x44k arrays

Four-day-old adult males were starved for 12 hours in a vial containing a filter paper impregnated with water before being transferred to caffeine-rich medium (18 mM; Sigma-Aldrich) for 12 hours. Food mixed with water served as a control. Taking into account the studies reported by Le Goff G (2006), and Willoughby L, (2006) [29 (link)–52 (link)], we decided to expose adult male flies for 12 hours on an 18 mM caffeine-rich medium to obtain a trade-off between the optimal induction of CYP genes and the lower mortality for the flies. For each food treatment (caffeine and water), RNA was isolated from 10 fly bodies (thorax and abdomen) using Isol RNA Lysis reagent (5Prime). All samples were prepared in triplicate to permit statistical analysis. Probe labeling, hybridization to single color Agilent 4x44k arrays, scanning and statistical analysis were performed by the IMAXIO company. Genes showing a 2-fold change and a significant p-value < 0.05 were considered to be differentially expressed. Microarray data obtained from this study can be accessed at NCBI GEO (GSE59084).

At specified times after amputation, plugs were extracted using 1 mm microcapillary pipets (FHC, catalog # 30-30-0, Bowdoin, ME), and transferred directly into Trizol (Life Technologies, Grand Island, NY) using a mouth pipet. For each replicate, 25 plugs were homogenized together, and then chloroform-extracted. The pellet was then precipitated with isopropanol, washed, and resuspended in water. RNA was then purified on an RNEasy column with DNase-treatment (Qiagen, Germany).
The experiment was performed in triplicate. RNA quality was assessed on a Bioanalyzer 2100 machine (Agilent, Santa Clara, CA). Starting with 100 ng total RNA, amplification and labeling with Cy3 or Cy5 was performed using the Low Input Quick Amp Labeling Kit Two-Color from Agilent Technologies (#5190-2306). Custom Agilent 4x44k arrays with design id: 033226 were hybridized according to the manufacturer protocols, and scanned on an Agilent G2505C scanner. Data was analyzed in the R environment using the Limma library (Smyth, 2004 (link)) for loess normalization and calculation of p-values between treatments. p-values were adjusted for multiple hypothesis testing by the method of (Benjamini and Hochberg, 1995 ). The data have been deposited in GEO with accession number: GSE56181.