Control cDNA microarray profiles of Drosophila head

In the process of developing a biological systems model of kindling-like plasticity in the fruit fly, we performed a control gene expression profiling analysis in which RNA isolated from different pools of heads of wild-type flies grown on normal food (NF) were compared between themselves using cDNA microarrays. Significance analysis of microarray (SAM) did not reveal any gene as differentially expressed below a false discovery rate (FDR) of 96.88%. Keywords: comparative D. melanogaster Oregon-R wild type flie were grown in standard fly medium, i.e., NF, consisting of agar-agar, maize powder, brown sugar, dried yeast and nipagin, at 24 + 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. To obtain flies used in the experiments, individuals from identical cultures were first allowed to lay eggs in milk bottles containing normal fly media. Flies were shifted to fresh bottle every 12 hr. First 4 sets of bottles were discarded. Flies that emerged in subsequent bottles were only used. Those that emerged in the beginning were first discarded and then flies were collected within 1 day, at 4 hr interval. Flies were anesthetized with diethyl ether, and males and females separated immediately after collection using a stereomicroscope. Male flies were thereafter maintained for another 3-4 days in standard culture vials containing NF in batches of 30 individuals. Flies were frozen in liquid nitrogen, shaken, and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, each representing flies collected from four vials two times of which comprised a single parallel set of treatment, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. The eight batches of purified cDNA was used for labeling with Cy3 and Cy5 dyes (Amersham Biosciences), four each, using Microarray RNA Target Synthesis Kit T7 (Roche) and labeled products were purified by Microarray Target Purification Kit (Roche). The two cRNA samples of each biological replicate, one labled with Cy3 and another with Cy5, were pooled together, precipitated, washed and air-dried. The dried pellet was dissolved in 18MΩ RNAase free water (Sigma). Hybridization solution was prepared by mixing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma) and added to the labeled product. This mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto). The slides were covered by lowering down a 24X60 mm coverslip (ESCO, Portsmouth, USA). Hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Coverslips were removed by submerging the slides in a solution containing 1X SSC and 0.1% SDS at 50ºC. Slides were washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Finally, slides were washed in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Microarray slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices), using both green and red lasers. The 16 bit TIFF images were preprocessed and quantified using Gene Pix Pro 6.0 software (Molecular Devices). Data normalization was performed using Acuity 4.0 software (Molecular Devices). Ratio based normalization was used for all slides. All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 2.21, Excel Add-In, Stanford) under the conditions of one class response and 100 permutations. SAM however did not reveal any gene as differentially expressed in NF versus NF below a false discovery rate (FDR) of 96.88%. It was therefore concluded that our experimental design is robust enough to reliably compare control versus treated samples next.