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| Status |
Public on Oct 04, 2007 |
| Title |
Effect of levetiracetam on gene expression in Drosophila head |
| Organism |
Drosophila melanogaster |
| Experiment type |
Expression profiling by array
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| Summary |
We recently developed a fly model of pentylenetetrazol (PTZ)-induced long-term behavioral plasticity. Pharmacological validation suggests this model to be kindling-like. Hence, our model is of relevance in understanding the molecular pathogenesis of epileptogenesis as well as in screening potential antiepileptogenic agents. Levetiracetam (LEV) is considered to have antiepileptogenic activity. Our fly model also predicted similar activity in LEV. Since the mechanism of action of LEV is not clearly known, we have examined the gene expression profiles of fly heads secondary to three days long chronic LEV treatment in Drosophila. Our results provide novel insights in to the drug’s mode of action.
Keywords: Drug Response
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| Overall design |
Oregon-R wild type D. melanogaster was grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin. Flies were cultured at 24 +/- 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Seven to ten days old unmated adult males were grown in either normal food (NF) or food containing 5 mg/ml of LEV for three days. Each culture vials contained 30 flies. Flies frozen in liquid nitrogen were agitated 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 LEV 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), and the cDNA purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of NF control and LEV 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 LEV 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 (D12Kv1, CDMC, Toronto). 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 16 bit TIFF images were preprocessed and quantified 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), and were detectable above background (SNR >= 2). 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 and 100 permutations
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| Contributor(s) |
Farhan M, Singh P, Sharma A |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Apr 10, 2007 |
| Last update date |
Mar 17, 2012 |
| Contact name |
Abhay Sharma |
| E-mail(s) |
abhaysharma@igib.res.in
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| Phone |
+91-11-27666156
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| Fax |
+91-11-27667602
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| Organization name |
CSIR-Institute of Genomics and Integrative Biology (IGIB)
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| Department |
Functional Genomics Unit
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| Street address |
Delhi University campus, Mall Road
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| City |
Delhi |
| State/province |
Delhi |
| ZIP/Postal code |
110007 |
| Country |
India |
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| Platforms (1) |
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| Samples (4)
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| GSM181438 |
NM-levetiracetam Replicate 4 (Dye Swap) |
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| Relations |
| BioProject |
PRJNA100307 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE7488_RAW.tar |
10.8 Mb |
(http)(custom) |
TAR (of GPR) |
| Processed data included within Sample table |
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