Project description: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 manufacturerM-bM-^@M-^Ys protocol. Double stranded cDNA was synthesized from 10 M-BM-5g of total RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturerM-bM-^@M-^Ys 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 18MM-NM-) 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 65M-BM-:C 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 37M-BM-:C for 16 hrs. Coverslips were removed by submerging the slides in a solution containing 1X SSC and 0.1% SDS at 50M-BM-:C. Slides were washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50M-BM-:C 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 10M-BM-5m 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.

Project description:Pentylenetetrazol (PTZ)-induced kindling in rodents is an established model of epileptogenesis. The molecular basis of the associated long-term plasticity is however not clear. Further, rodent models of kindling plasticity are not amenable to large-scale screening of compounds for identifying antiepileptogenic drugs. In this context, we have developed a fly model of chronic PTZ- and withdrawal-induced behavioral plasticity. In our model, the chronic treatment is given for 7 days, followed by 7 day long withdrawal. Gene expression profiles of fly heads secondary to chronic PTZ were examined to identify molecular correlates of behavior in our model. Expression profiling of fly heads at three time points in the 7 day long chronic PTZ - 12 hrs, 2nd day, and 7th day - showed a dynamic and widespread alteration of various functional categories of genes. Keywords: Time Series 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 of PTZ for seven days. Following this, the control and PTZ 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 three time points during PTZ treatement - 12hrs, 2nd day, and 7th day. 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 PTZ exposed individuals, during treatment or withdrawal period, using TRI REAGENT (Sigma) according to the manufacturerM-bM-^@M-^Ys protocol. Double stranded cDNA was synthesized from 10 M-BM-5g of total RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturerM-bM-^@M-^Ys 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 18MM-NM-) 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 65M-BM-:C 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 37M-BM-:C for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50M-BM-:C, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50M-BM-:C 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 10M-BM-5m 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.

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.

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 ÂºC, 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 Âµg 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 18MÎ© 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 65ÂºC 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 37ÂºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ÂºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ÂºC 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 10Âµm 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.

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Control cDNA microarray profiles of Drosophila head

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