Gene expression changes in Drosophila head induced by pentylenetetrazole and levetiracetam combination treatment
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
SUBMITTER: Abhay Sharma
PROVIDER: E-GEOD-10850 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Drosophila melanogaster adult flies fed on normal food (NF) containing 16 mg/ml of pentylenetetrazole (PTZ) in the food show a hyperkinetic behavior within 24 hours. Half of that concentration, i.e., 8 mg/ml, of the PTZ, if fed for seven days, though doesn’t cause seizure-like behavior, results in a decreased climbing speed in flies. This change in locomotor behavior is progressive and becomes significant only on seventh day of the treatment. This climbing deficit is ameliorated when flies are treated concomitantly with PTZ and either of the two antiepileptic drugs (AEDs), sodium valproate (NaVP) or levetiracetam (LEV). Further experiments based on different regimes of combination and singular PTZ treatment demonstrated that whereas NaVP show a weaker prophylactic and stronger symptomatic effect, LEV exhibit an opposite effect, i.e., stronger prophylactic and weaker symptomatic action. These observations are consistent with the therapeutic effect of antiepileptic drugs. Time series of microarray gene expression profiling (earlier GEO submission) during chronic PTZ provided evidence of underlying downregulation of three main categories of biological processes, synaptic remodeling, energy metabolism and transport, in that order, in fly head. Transcriptomic analysis secondary to NaVP and LEV (earlier GEO submission) was found to cause statistically significant upregulation of two biological process categories each, energy metabolism and transport in case of NaVP and synaptic remodeling and energy metabolism in case of LEV. Time series of genome wide expression profiling secondary to PTZ and LEV combination treatment (earlier GEO submission) showed neutralizing effect of LEV on PTZ induced expression changes. Mining of published transcriptomic and proteomic data pertaining to epilepsy or established rodent animal models of epileptogenesis supported the relevance of the fly model in understanding the underlying brain pathophysiology. Systems modeling using RNA interference and small molecules demonstrated the robustness of the fly model. In brief, the above results clearly showed that the fly model is valuable not only in screening of antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents, but also in advancing our knowledge of mechanisms of action of drugs used in treating human patients suffering from various neurological and neuropsychiatric conditions, pharmacogenomics of these drugs, identification of potential drug targets, and selection of potential candidate genes for association analysis in epilepsy. Considering the above fly locomotor behavior model as relevant in kindling attainment, it was of obvious interest to study the behavioral and transcriptomic changes post-kindling, i.e., after withdrawing PTZ following seven day long chronic PTZ administration. It was observed that when PTZ is discontinued and flies climbing speed is monitored for next seven days of PTZ discontinuation, climbing speed deficit initially disappears and then a progressive increase in climbing speed is detected. Behavioral pharmacology of AEDs in post-kindling regime, like fly kindling described above, showed its usefulness in screening of potential antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents. The present submission relates to transcriptomic changes in fly head secondary to levetiracetam (LEV) treatment following discontinuation of PTZ for seven days (after seven days of chronic PTZ treatment). To delineate possible prophylactic or symptomatic effect of LEV at behavioral level, flies were treated with LEV for first three days and then shifted to NF for next four days (3 + 4 day regime) or flies treated with NF for first four days and then drug treated for next three days (4 + 3 day regime), in that order. At transcriptomic level, we have now examined the effect of LEV in 3 + 4 day regime. Gene expression profiles have been generated at both time points, 3rd and 7th day, i.e., on 10th and 14th day from the beginning of PTZ treatment. Effect of three day long chronic LEV treatment on fly head microarray gene expression profile, in otherwise normally grown flies, has already been determined (earlier GEO submission). Similarly, transcriptomic changes after one day, three days and seven days of PTZ discontinuation (i.e., 8th, 10th and 14th day of the beginning of chronic PTZ treatment) have already been deciphered (earlier GEO submission). Unlike the previous experiment in which flies were maintained in the same container during the post-kindling period of seven days, flies needed to be shifted in fresh vials once in the present experiment because of the demand of 3 + 4 day regime. Since change in housing conditions has the potential to influence head transcriptome, we have also generated microarray expression profile of fly head after treating flies with PTZ for seven days, maintaining the flies to NF containing vials for three days and then shifting and maintaining them in fresh NF containing vials for next four days (submitted in GEO previously). Keywords: Drug Response 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 + 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in either normal food (NF) or food containing 8 mg/ml PTZ for seven days. Each treatment vial contained 30 flies in the beginning. Next, flies were treated in either of the following two manners: (1) shifted to vials containing 5 mg/ml LEV media for three days, and (2) shifted to LEV media for three days and then again shifted to fresh vials containing NF for next four days. Heads were harvested at the end of either of the two treatment conditions. 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 individuals treated in either one of the two ways described earlier, 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 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), 10 mg/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, 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 10 µm 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, Excel Add-In, Stanford) under the conditions of one class response, imputation and 100 permutations.
2008-05-03 | E-GEOD-10988 | biostudies-arrayexpress
Project description:Kindling induced by Pentylenetetrazol is an established rodent model of epileptogenesis. The molecular basis of the long-term plasticity involved is however not clear. In addition, rodent models of kindling plasticity are not useful for large-scale screening of compounds to identify antiepileptogenic drugs. Given this, we have developed a fly model of chronic PTZ- and withdrawal-induced behavioral plasticity. In our fly model, the chronic PTZ treatment is given for 7 days. This is then followed by 7 day long withdrawal. Expression profiling of fly heads at three time points in the 7 day long withdrawal period – 8th day, 10th day, and 14th day from the beginning of the treatment - showed a dynamic and widespread alteration of various functional categories of genes. D. melanogaster Oregon-R wild type cultures were grown at 24 + 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old adult males, unmated, were grown in either normal food (NF) or food containing 8 mg/ml of PTZ for seven days. Following this, the control and PTZ treated individuals were shifted to vials containing NF and maintained further for seven days. Each vial contained 30 individuals in the beginning of the treatment. Heads were harvested at three time points during withdrawal - 8th day, 10th day, and 14th day from the beginning of the treatment. Heads of flies frozen in liquid nitrogen were collected, after vigorous shaking of the vial containing the flies, 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 PTZ exposed individuals, during treatment or withdrawal period. For isolation, TRI REAGENT (Sigma) was used as per manufacturer’s recommendation. 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 PTZ 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 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 2.21, Excel Add-In, Stanford) under the conditions of one class response and 100 permutations.
2007-10-01 | E-GEOD-7156 | biostudies-arrayexpress
Project description:Drosophila melanogaster adult flies fed on normal food (NF) containing 16 mg/ml of pentylenetetrazole (PTZ) in the food show a hyperkinetic behavior within 24 hours. Half of that concentration, i.e., 8 mg/ml, of the PTZ, if fed for seven days, though doesn’t cause seizure-like behavior, results in a decreased climbing speed in flies. This change in locomotor behavior is progressive and becomes significant only on seventh day of the treatment. This climbing deficit is ameliorated when flies are treated concomitantly with PTZ and either of the two antiepileptic drugs (AEDs), sodium valproate (NaVP) or levetiracetam (LEV). Further experiments based on different regimes of combination and singular PTZ treatment demonstrated that whereas NaVP show a weaker prophylactic and stronger symptomatic effect, LEV exhibit an opposite effect, i.e., stronger prophylactic and weaker symptomatic action. These observations are consistent with the therapeutic effect of antiepileptic drugs. Time series of microarray gene expression profiling (earlier GEO submission) during chronic PTZ provided evidence of underlying downregulation of three main categories of biological processes, synaptic remodeling, energy metabolism and transport, in that order, in fly head. Transcriptomic analysis secondary to NaVP and LEV (earlier GEO submission) was found to cause statistically significant upregulation of two biological process categories each, energy metabolism and transport in case of NaVP and synaptic remodeling and energy metabolism in case of LEV. Time series of genome wide expression profiling secondary to PTZ and LEV combination treatment (earlier GEO submission) showed neutralizing effect of LEV on PTZ induced expression changes. Mining of published transcriptomic and proteomic data pertaining to epilepsy or established rodent animal models of epileptogenesis supported the relevance of the fly model in understanding the underlying brain pathophysiology. Systems modeling using RNA interference and small molecules demonstrated the robustness of the fly model. In brief, the above results clearly showed that the fly model is valuable not only in screening of antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents, but also in advancing our knowledge of mechanisms of action of drugs used in treating human patients suffering from various neurological and neuropsychiatric conditions, pharmacogenomics of these drugs, identification of potential drug targets, and selection of potential candidate genes for association analysis in epilepsy. Considering the above fly locomotor behavior model as relevant in kindling attainment, it was of obvious interest to study the behavioral and transcriptomic changes post-kindling, i.e., after withdrawing PTZ following seven day long chronic PTZ administration. It was observed that when PTZ is discontinued and flies climbing speed is monitored for next seven days of PTZ discontinuation, climbing speed deficit initially disappears and then a progressive increase in climbing speed is detected. Behavioral pharmacology of AEDs in post-kindling regime, like fly kindling described above, showed its usefulness in screening of potential antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents. The present submission relates to transcriptomic changes in fly head secondary to ethosuximide (ETH) treatment following discontinuation of PTZ for seven days (after seven days of chronic PTZ treatment). To delineate possible prophylactic or symptomatic effect of ETH at behavioral level, flies were treated with ETH for first three days and then shifted to NF for next four days (3 + 4 day regime) or flies treated with NF for first four days and then drug treated for next three days (4 + 3 day regime), in that order. At transcriptomic level, we have now examined the effect of ETH in 3 + 4 day regime. Gene expression profiles have been generated at both time points, 3rd and 7th day, i.e., on 10th and 14th day from the beginning of PTZ treatment. Effect of three day long chronic ETH treatment on fly head microarray gene expression profile, in otherwise normally grown flies, has already been determined (earlier GEO submission). Similarly, transcriptomic changes after one day, three days and seven days of PTZ discontinuation (i.e., 8th, 10th and 14th day of the beginning of chronic PTZ treatment) have already been deciphered (earlier GEO submission). However, unlike the previous experiment in which flies were maintained in the same container during the post-kindling period of seven days, flies needed to be shifted in fresh vials once in the present experiment because of the demand of 3 + 4 day regime. Since change in housing conditions has the potential to influence head transcriptome, we are also submitting here microarray expression profile of fly head after treating flies with PTZ for seven days, maintaining the flies to NF containing vials for three days and then shifting and maintaining them in fresh NF containing vials for next four days. Repetition of expression profiling on 3rd day of withdrawal (i.e., 10th day from beginning of PTZ treatment) was not needed because both the previous and the present experiment were similar with respect to housing conditions. Repetition of expression profiling on 7th day (i.e., 14th day from the beginning of PTZ treatment) was however required in view of change in housing conditions. The present submission therefore includes this later expression profile also. 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 + 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in either normal food (NF) or food containing 8 mg/ml PTZ for seven days. Each treatment vial contained 30 flies in the beginning. Next, flies were treated in either of the following manner: (1) shifted to vials containing 3.48 mg/ml ETH media for three days, (2) shifted to ETH media for three days and then again shifted to fresh vials containing NF for next four days, and (3) shifted to NF vials for three days and then again shifted to fresh NF vial for the next four days. Heads were harvested at the end of either of the three treatment conditions. 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 individuals treated in either one of the three ways described earlier, 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 drug 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 10 mg/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, 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 10 µm 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, Excel Add-In, Stanford) under the conditions of one class response, imputation and 100 permutations.
2008-05-03 | E-GEOD-10984 | biostudies-arrayexpress
Project description:Drosophila melanogaster adult flies fed on normal food (NF) containing 16 mg/ml of pentylenetetrazole (PTZ) in the food show a hyperkinetic behavior within 24 hours. Half of that concentration, i.e., 8 mg/ml, of the PTZ, if fed for seven days, though doesn’t cause seizure-like behavior, results in a decreased climbing speed in flies. This change in locomotor behavior is progressive and becomes significant only on seventh day of the treatment. This climbing deficit is ameliorated when flies are treated concomitantly with PTZ and either of the two antiepileptic drugs (AEDs), sodium valproate (NaVP) or levetiracetam (LEV). Further experiments based on different regimes of combination and singular PTZ treatment demonstrated that whereas NaVP show a weaker prophylactic and stronger symptomatic effect, LEV exhibit an opposite effect, i.e., stronger prophylactic and weaker symptomatic action. These observations are consistent with the therapeutic effect of antiepileptic drugs. Time series of microarray gene expression profiling (earlier GEO submission) during chronic PTZ provided evidence of underlying downregulation of three main categories of biological processes, synaptic remodeling, energy metabolism and transport, in that order, in fly head. Transcriptomic analysis secondary to NaVP and LEV (earlier GEO submission) was found to cause statistically significant upregulation of two biological process categories each, energy metabolism and transport in case of NaVP and synaptic remodeling and energy metabolism in case of LEV. Time series of genome wide expression profiling secondary to PTZ and LEV combination treatment (earlier GEO submission) showed neutralizing effect of LEV on PTZ induced expression changes. Mining of published transcriptomic and proteomic data pertaining to epilepsy or established rodent animal models of epileptogenesis supported the relevance of the fly model in understanding the underlying brain pathophysiology. Systems modeling using RNA interference and small molecules demonstrated the robustness of the fly model. In brief, the above results clearly showed that the fly model is valuable not only in screening of antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents, but also in advancing our knowledge of mechanisms of action of drugs used in treating human patients suffering from various neurological and neuropsychiatric conditions, pharmacogenomics of these drugs, identification of potential drug targets, and selection of potential candidate genes for association analysis in epilepsy. Considering the above fly locomotor behavior model as relevant in kindling attainment, it was of obvious interest to study the behavioral and transcriptomic changes post-kindling, i.e., after withdrawing PTZ following seven day long chronic PTZ administration. It was observed that when PTZ is discontinued and flies climbing speed is monitored for next seven days of PTZ discontinuation, climbing speed deficit initially disappears and then a progressive increase in climbing speed is detected. Behavioral pharmacology of AEDs in post-kindling regime, like fly kindling described above, showed its usefulness in screening of potential antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents. The present submission relates to transcriptomic changes in fly head secondary to gabapentin (GBP) treatment following discontinuation of PTZ for seven days (after seven days of chronic PTZ treatment). To delineate possible prophylactic or symptomatic effect of GBP at behavioral level, flies were treated with GBP for first three days and then shifted to NF for next four days (3 + 4 day regime) or flies treated with NF for first four days and then drug treated for next three days (4 + 3 day regime), in that order. At transcriptomic level, we have now examined the effect of GBP in 3 + 4 day regime. Gene expression profiles have been generated at both time points, 3rd and 7th day, i.e., on 10th and 14th day from the beginning of PTZ treatment. Effect of three day long chronic GBP treatment on fly head microarray gene expression profile, in otherwise normally grown flies, has already been determined (earlier GEO submission). Similarly, transcriptomic changes after one day, three days and seven days of PTZ discontinuation (i.e., 8th, 10th and 14th day of the beginning of chronic PTZ treatment) have already been deciphered (earlier GEO submission). Unlike the previous experiment in which flies were maintained in the same container during the post-kindling period of seven days, flies needed to be shifted in fresh vials once in the present experiment because of the demand of 3 + 4 day regime. Since change in housing conditions has the potential to influence head transcriptome, we have also generated microarray expression profile of fly head after treating flies with PTZ for seven days, maintaining the flies to NF containing vials for three days and then shifting and maintaining them in fresh NF containing vials for next four days (submitted in GEO previously). 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 + 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in either normal food (NF) or food containing 8 mg/ml PTZ for seven days. Each treatment vial contained 30 flies in the beginning. Next, flies were treated in either of the following two manners: (1) shifted to vials containing 16 mg/ml GBP media for three days, and (2) shifted to GBP media for three days and then again shifted to fresh vials containing NF for next four days. Heads were harvested at the end of either of the two treatment conditions. 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 individuals treated in either one of the two ways described earlier, 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 GBP 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), 10 mg/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, 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 10 µm 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, Excel Add-In, Stanford) under the conditions of one class response, imputation and 100 permutations.
2008-05-03 | E-GEOD-10985 | biostudies-arrayexpress
Project description:Pilocarpine hydrochloride (PILO) is an epileptogenic agent. In an effort to develop a Drosophila model of kindling epileptogenesis, we have examined chronic PILO induced alterations in gene expression profile of fly heads. Expression profiling of fly heads after 7 day long PILO treatment revealed perturbations in various functional category of genes. Keywords: Drug Response 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 +/- 1ºC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in either normal food (NF) or food containing 1.5 mg/ml of PILO for seven days. Following this, the control and PILO treated flies were shifted to vials containing normal food and maintained further for seven days. Each vial contained 30 flies in the beginning of the treatment. Heads were harvested at the end of seven day treatment. 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 PILO exposed individuals, during treatment or withdrawal period, using TRI REAGENT (Sigma) according to the manufacturerâs 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 manufacturerâs 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 PILO 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 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 and 100 permutations.
2010-05-25 | E-GEOD-7787 | biostudies-arrayexpress
Project description:Drosophila melanogaster adult flies fed on normal food (NF) containing 16 mg/ml of pentylenetetrazole (PTZ) in the food show a hyperkinetic behavior within 24 hours. Half of that concentration, i.e., 8 mg/ml, of the PTZ, if fed for seven days, though doesn’t cause seizure-like behavior, results in a decreased climbing speed in flies. This change in locomotor behavior is progressive and becomes significant only on seventh day of the treatment. This climbing deficit is ameliorated when flies are treated concomitantly with PTZ and either of the two antiepileptic drugs (AEDs), sodium valproate (NaVP) or levetiracetam (LEV). Further experiments based on different regimes of combination and singular PTZ treatment demonstrated that whereas NaVP show a weaker prophylactic and stronger symptomatic effect, LEV exhibit an opposite effect, i.e., stronger prophylactic and weaker symptomatic action. These observations are consistent with the therapeutic effect of antiepileptic drugs. Time series of microarray gene expression profiling (earlier GEO submission) during chronic PTZ provided evidence of underlying downregulation of three main categories of biological processes, synaptic remodeling, energy metabolism and transport, in that order, in fly head. Transcriptomic analysis secondary to NaVP and LEV (earlier GEO submission) was found to cause statistically significant upregulation of two biological process categories each, energy metabolism and transport in case of NaVP and synaptic remodeling and energy metabolism in case of LEV. Time series of genome wide expression profiling secondary to PTZ and LEV combination treatment (earlier GEO submission) showed neutralizing effect of LEV on PTZ induced expression changes. Mining of published transcriptomic and proteomic data pertaining to epilepsy or established rodent animal models of epileptogenesis supported the relevance of the fly model in understanding the underlying brain pathophysiology. Systems modeling using RNA interference and small molecules demonstrated the robustness of the fly model. In brief, the above results clearly showed that the fly model is valuable not only in screening of antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents, but also in advancing our knowledge of mechanisms of action of drugs used in treating human patients suffering from various neurological and neuropsychiatric conditions, pharmacogenomics of these drugs, identification of potential drug targets, and selection of potential candidate genes for association analysis in epilepsy. Considering the above fly locomotor behavior model as relevant in kindling attainment, it was of obvious interest to study the behavioral and transcriptomic changes post-kindling, i.e., after withdrawing PTZ following seven day long chronic PTZ administration. It was observed that when PTZ is discontinued and flies climbing speed is monitored for next seven days of PTZ discontinuation, climbing speed deficit initially disappears and then a progressive increase in climbing speed is detected. Behavioral pharmacology of AEDs in post-kindling regime, like fly kindling described above, showed its usefulness in screening of potential antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents. The present submission relates to transcriptomic changes in fly head secondary to NaVP treatment following discontinuation of PTZ for seven days (after seven days of chronic PTZ treatment). To delineate possible prophylactic or symptomatic effect of NaVP at behavioral level, flies were treated with NaVP for first three days and then shifted to NF for next four days (3 + 4 day regime) or flies treated with NF for first four days and then drug treated for next three days (4 + 3 day regime), in that order. At transcriptomic level, we have now examined the effect of NaVP in 3 + 4 day regime. Gene expression profiles have been generated at both time points, 3rd and 7th day, i.e., on 10th and 14th day from the beginning of PTZ treatment. Effect of three day long chronic NaVP treatment on fly head microarray gene expression profile, in otherwise normally grown flies, has already been determined (earlier GEO submission). Similarly, transcriptomic changes after one day, three days and seven days of PTZ discontinuation (i.e., 8th, 10th and 14th day of the beginning of chronic PTZ treatment) have already been deciphered (earlier GEO submission). Unlike the previous experiment in which flies were maintained in the same container during the post-kindling period of seven days, flies needed to be shifted in fresh vials once in the present experiment because of the demand of 3 + 4 day regime. Since change in housing conditions has the potential to influence head transcriptome, we have also generated microarray expression profile of fly head after treating flies with PTZ for seven days, maintaining the flies to NF containing vials for three days and then shifting and maintaining them in fresh NF containing vials for next four days (submitted in GEO previously). 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 + 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in either normal food (NF) or food containing 0.33 mg/ml PTZ for seven days. Each treatment vial contained 30 flies in the beginning. Next, flies were treated in either of the following two manners: (1) shifted to vials containing 24 mg/ml NaVP media for three days, and (2) shifted to NaVP media for three days and then again shifted to fresh vials containing NF for next four days. Heads were harvested at the end of either of the two treatment conditions. 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 individuals treated in either one of the two ways described earlier, 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 18M? RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and NaVP 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), 10 mg/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, 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 10 µm 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, Excel Add-In, Stanford) under the conditions of one class response, imputation and 100 permutations.
2008-05-03 | E-GEOD-10986 | biostudies-arrayexpress
Project description:Drosophila melanogaster adult flies fed on normal food (NF) containing 16 mg/ml of pentylenetetrazole (PTZ) in the food show a hyperkinetic behavior within 24 hours. Half of that concentration, i.e., 8 mg/ml, of the PTZ, if fed for seven days, though doesn’t cause seizure-like behavior, results in a decreased climbing speed in flies. This change in locomotor behavior is progressive and becomes significant only on seventh day of the treatment. This climbing deficit is ameliorated when flies are treated concomitantly with PTZ and either of the two antiepileptic drugs (AEDs), sodium valproate (NaVP) or levetiracetam (LEV). Further experiments based on different regimes of combination and singular PTZ treatment demonstrated that whereas NaVP show a weaker prophylactic and stronger symptomatic effect, LEV exhibit an opposite effect, i.e., stronger prophylactic and weaker symptomatic action. These observations are consistent with the therapeutic effect of antiepileptic drugs. Time series of microarray gene expression profiling (earlier GEO submission) during chronic PTZ provided evidence of underlying downregulation of three main categories of biological processes, synaptic remodeling, energy metabolism and transport, in that order, in fly head. Transcriptomic analysis secondary to NaVP and LEV (earlier GEO submission) was found to cause statistically significant upregulation of two biological process categories each, energy metabolism and transport in case of NaVP and synaptic remodeling and energy metabolism in case of LEV. Time series of genome wide expression profiling secondary to PTZ and LEV combination treatment (earlier GEO submission) showed neutralizing effect of LEV on PTZ induced expression changes. Mining of published transcriptomic and proteomic data pertaining to epilepsy or established rodent animal models of epileptogenesis supported the relevance of the fly model in understanding the underlying brain pathophysiology. Systems modeling using RNA interference and small molecules demonstrated the robustness of the fly model. In brief, the above results clearly showed that the fly model is valuable not only in screening of antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents, but also in advancing our knowledge of mechanisms of action of drugs used in treating human patients suffering from various neurological and neuropsychiatric conditions, pharmacogenomics of these drugs, identification of potential drug targets, and selection of potential candidate genes for association analysis in epilepsy. Considering the above fly locomotor behavior model as relevant in kindling attainment, it was of obvious interest to study the behavioral and transcriptomic changes post-kindling, i.e., after withdrawing PTZ following seven day long chronic PTZ administration. It was observed that when PTZ is discontinued and flies climbing speed is monitored for next seven days of PTZ discontinuation, climbing speed deficit initially disappears and then a progressive increase in climbing speed is detected. Behavioral pharmacology of AEDs in post-kindling regime, like fly kindling described above, showed its usefulness in screening of potential antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents. The present submission relates to transcriptomic changes in fly head secondary to vigabatrin (VGB) treatment following discontinuation of PTZ for seven days (after seven days of chronic PTZ treatment). To delineate possible prophylactic or symptomatic effect of VGB at behavioral level, flies were treated with VGB for first three days and then shifted to NF for next four days (3 + 4 day regime) or flies treated with NF for first four days and then drug treated for next three days (4 + 3 day regime), in that order. At transcriptomic level, we have now examined the effect of VGB in 3 + 4 day regime. Gene expression profiles have been generated at both time points, 3rd and 7th day, i.e., on 10th and 14th day from the beginning of PTZ treatment. Effect of three day long chronic VGB treatment on fly head microarray gene expression profile, in otherwise normally grown flies, has already been determined (earlier GEO submission). Similarly, transcriptomic changes after one day, three days and seven days of PTZ discontinuation (i.e., 8th, 10th and 14th day of the beginning of chronic PTZ treatment) have already been deciphered (earlier GEO submission). Unlike the previous experiment in which flies were maintained in the same container during the post-kindling period of seven days, flies needed to be shifted in fresh vials once in the present experiment because of the demand of 3 + 4 day regime. Since change in housing conditions has the potential to influence head transcriptome, we have also generated microarray expression profile of fly head after treating flies with PTZ for seven days, maintaining the flies to NF containing vials for three days and then shifting and maintaining them in fresh NF containing vials for next four days (submitted in GEO previously). 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 + 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in either normal food (NF) or food containing 8 mg/ml PTZ for seven days. Each treatment vial contained 30 flies in the beginning. Next, flies were treated in either of the following two manners: (1) shifted to vials containing 24 mg/ml VGB media for three days, and (2) shifted to VGB media for three days and then again shifted to fresh vials containing NF for next four days. Heads were harvested at the end of either of the two treatment conditions. 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 individuals treated in either one of the two ways described earlier, 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 VGB 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), 10 mg/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, 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 10 µm 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, Excel Add-In, Stanford) under the conditions of one class response, imputation and 100 permutations.
2008-05-03 | E-GEOD-10987 | biostudies-arrayexpress
Project description:Tetraethylammonium chloride (TEA) is an epileptogenic agent. In an effort to develop a Drosophila model of kindling epileptogenesis, we have examined chronic TEA induced alterations in gene expression profile of fly heads. Expression profiling of fly heads after 7 day long TEA treatment revealed perturbations in various functional category of genes. Keywords: Drug Response 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 ºC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in either normal food (NF) or food containing 16 mg/ml of TEA for seven days. Following this, the control and TEA treated flies were shifted to vials containing normal food and maintained further for seven days. Each vial contained 30 flies in the beginning of the treatment. Heads were harvested at the end of seven day treatment. 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 TEA exposed individuals, during treatment or withdrawal period, using TRI REAGENT (Sigma) according to the manufacturerâs 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 manufacturerâs 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 TEA 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 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 and 100 permutations.
2010-05-25 | E-GEOD-7783 | biostudies-arrayexpress
Project description:Strychnine (STR) is a convulsant agent. In an effort to develop a Drosophila model of kindling epileptogenesis, we have examined chronic STR induced alterations in gene expression profile of fly heads. Expression profiling of fly heads after 7 day long STR treatment revealed perturbations in various functional category of genes. Keywords: Drug Response 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 ºC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in either normal food (NF) or food containing 3.33 mg/ml of STR for seven days. Following this, the control and STR treated flies were shifted to vials containing normal food and maintained further for seven days. Each vial contained 30 flies in the beginning of the treatment. Heads were harvested at the end of seven day treatment. 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 STR exposed individuals, during treatment or withdrawal period, using TRI REAGENT (Sigma) according to the manufacturerâs 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 manufacturerâs 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 STR 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 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 and 100 permutations.
2010-05-25 | E-GEOD-7786 | biostudies-arrayexpress
Project description: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 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 manufacturerâs 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 manufacturerâs 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.
2010-05-25 | E-GEOD-10808 | biostudies-arrayexpress