|
| Status |
Public on Mar 26, 2009 |
| Title |
F2 male CNS-3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Drosophila melanogaster, males, unmated
|
| Organism |
Drosophila melanogaster |
| Characteristics |
generation: F2
strain: Oregon R
sex: male
tissue: head
|
| Biomaterial provider |
Institute of Genomics and Integrative Biology (IGIB)
|
| Treatment protocol |
Male flies from F2 generation (control) were allowed to grow on normal media till 3-4 days old, and then heads collected and stored at –70ºC.
|
| Growth protocol |
D. melanogaster Oregon-R wild type male flies of F0 generation which were kept in normal food for 7 days and again shifted to normal food for next 7 days, were allowed to mate with female flies(never treated with drug). After 2 days parents were removed and eggs were allowed to grow on standard fly medium.F1 progeny was collected, males and females were separated, and after 3-4 days were kept for crossing in normal food vials and removed after 2 days. F2 progeny thus emerged was collected and used for experiments after 3-4 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from head using TRI REAGENT (Sigma) according to the manufacturer’s protocol.
|
| Label |
Cy5
|
| Label protocol |
Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche) and purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. This cDNA was used for synthesizing labeled cRNA with Cy5 dye (Amersham Biosciences),using Microarray RNA Target Synthesis Kit T7 (Roche) and labeled cRNA was purified by Microarray Target Purification Kit (Roche).
|
| |
|
| Channel 2 |
| Source name |
Drosophila melanogaster, males, unmated (F0 were PTZ-treated)
|
| Organism |
Drosophila melanogaster |
| Characteristics |
generation: F2
strain: Oregon R
sex: male
tissue: head
|
| Biomaterial provider |
Institute of Genomics and Integrative Biology (IGIB)
|
| Treatment protocol |
Male flies from F2 generation (treatment) were allowed to grow on normal media till 3-4 days old, and then heads collected and stored at –70ºC.
|
| Growth protocol |
D. melanogaster Oregon-R wild type male flies of F0 generation which were kept in PTZ food for 7 days and again shifted to normal food for next 7 days, were allowed to mate with female flies(never treated with drug). After 2 days parents were removed and eggs were allowed to grow on standard fly medium.F1 progeny was collected, males and females were separated, and after 3-4 days were kept for crossing in normal food vials and removed after 2 days. F2 progeny thus emerged was collected and used for experiments after 3-4 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from head using TRI REAGENT (Sigma) according to the manufacturer’s protocol.
|
| Label |
Cy3
|
| Label protocol |
Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche) and purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. This cDNA was used for synthesizing labeled cRNA with Cy3 dye (Amersham Biosciences),using Microarray RNA Target Synthesis Kit T7 (Roche) and labeled cRNA was purified by Microarray Target Purification Kit (Roche).
|
| |
|
| |
| Hybridization protocol |
The two cRNA samples of each biological replicate, one labled with Cy3 and another with Cy5, were pooled together, precipitated, washed and air-dried. The dried pellet was dissolved in 18MΩ RNAase free water (Sigma). Hybridization solution was prepared by mixing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma) and added to the labeled product. This mixture was denatured at 65ºC and applied onto cDNA microarray slides. The slides were covered by lowering down a 24X60 mm coverslip (ESCO, Portsmouth, USA). Hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs
|
| Scan protocol |
Microarray slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices), using both green and red lasers. The 16 bit TIFF images were preprocessed and quantified using Gene Pix Pro 6.0 software (Molecular Devices).
|
| Description |
F2 male CNS-3
|
| Data processing |
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), 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.
|
| |
|
| Submission date |
Mar 05, 2009 |
| Last update date |
Mar 25, 2009 |
| Contact name |
Abhay Sharma |
| E-mail(s) |
[email protected]
|
| Phone |
+91-11-27666156
|
| Fax |
+91-11-27667602
|
| Organization name |
CSIR-Institute of Genomics and Integrative Biology (IGIB)
|
| Department |
Functional Genomics Unit
|
| Street address |
Delhi University campus, Mall Road
|
| City |
Delhi |
| State/province |
Delhi |
| ZIP/Postal code |
110007 |
| Country |
India |
| |
|
| Platform ID |
GPL3603 |
| Series (1) |
| GSE15136 |
A systematic search for transgenerational effect of drug exposure to adult males using a novel Drosophila model |
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