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Chronic potassium chloride (KCl) treatment induced gene expression changes in Drosophila head

ABSTRACT: We have recently developed a Drosophila behavioral- and transcriptomic- based (systems) model of chronic pentylenetetrazole induced locomotor plasticity. Pharmacological validation at behavioral level and at the level of fly head microarray gene expression shows that the model is of potential value in screening of compounds for detecting antiepileptic, antiepileptogenic, disease-modifying and neuroprotective activities. While screening compounds using this model at behavioral level, potassium chloride (KCl) was identified as having therapeutic potential. Examination of transcriptomic changes induced by KCl in fly head further supports its potential use as a therapeutic agent in neurological and neuropsychiatric conditions. Keywords: Drug Response Overall design: D. melanogaster Oregon-R wild type flies were grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin. The cultures were grown at 24 + 1 C, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Ten to eleven days old unmated adult males were grown in either normal food (NF) or food containing 100 mM KCl for three days. Each treatment vial contained 30 flies. Heads were harvested at the end of seven day treatment. For this, flies frozen in liquid nitrogen were shaken and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four KCl treated individuals, 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. Each of the four sets of control and treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and KCl treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing 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). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D14Kv1, CDMC, Toronto, Canada). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides 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). Slides were given a final wash 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. Slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The preprocessing and quantification of the 16 bit TIFF images were carried out using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). 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 the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 3.0, Excel Add-In, Stanford) under the conditions of one class response, imputation and 100 permutations.

INSTRUMENT(S): CDMC_Drosophila_Oligo14kv1/Oligo14kv2

ORGANISM(S): Drosophila melanogaster  

SUBMITTER: Abhay Sharma  

PROVIDER: GSE10808 | GEO | 2008-03-18



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