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Sample GSM285838 Query DataSets for GSM285838
Status Public on Feb 11, 2009
Title tud progeny female 72 hr APF - replicate 1
Sample type RNA
 
Channel 1
Source name tud progeny female 72 hr APF
Organism Drosophila melanogaster
Characteristics Strain: tud Progeny
Gender: Female
Age: 72 hr APF
Tissue: Whole Body
Treatment protocol Female tud progeny were collected at the white pre-pupal stage between Zeitgeber time (ZT) 1 and ZT 4, aged at 25◦C for 72 hours and then snap frozen in liquid nitrogen.
Extracted molecule total RNA
Extraction protocol RNA was isolated from ~30 pupae by homogenization and extraction using the TRIzol® protocol (Invitrogen, Carlsbad, CA) and resuspended in 20µL diethylpyrocarbonate (DEPC)-treated H2O.
Label Cy5
Label protocol cDNAs were directly labeled with Cy5 or Cy3 during the reverse transcription reaction; 30µg of total RNA was used as a starting template. The reverse transcription reaction was performed for two hours at 42◦C using the following reagents (values in parentheses are final molarity or final concentration): oligo dT primer (Operon, 3.75 μM), dithiothreitol (Invitrogen, 10 mM), First Strand Buffer (Invitrogen, 1×), dNTPs minus dTTP (Invitrogen, 0.5 mM), dTTP (Invitrogen, 50 μM), Cy-labeled dUTP (Perkin-Elmer, 0.625 nM), and Superscript II reverse transcriptase (Invitrogen, 10 U/μL). The reaction was stopped and RNA was hydrolyzed by a 20 minute incubation at 65◦C with NaOH (167 mM) and EDTA (83 mM). After neutralizing by adding HEPES buffer (pH8.0; 294 mM) and sodium acetate (pH5.2; 228 mM), cDNA samples were purified (Gel-Purification Kit, Qiagen, Valencia, CA).
 
Channel 2
Source name Reference
Organism Drosophila melanogaster
Characteristics Strain: Canton S
Gender: Male and Female
Age: All pupal stages
Tissue: Whole Body
Treatment protocol Male and female flies of all pupal stages were collected and snap frozen in liquid nitrogen.
Extracted molecule total RNA
Extraction protocol RNA was isolated from the entire batch by homogenization and extraction using the TRIzol(R) protocol (Invitrogen, Carlsbad, CA) and 30 ug were resuspended in 20µL diethylpyrocarbonate (DEPC)-treated H2O.
Label Cy3
Label protocol cDNAs were directly labeled with Cy5 or Cy3 during the reverse transcription reaction; 30µg of total RNA was used as a starting template. The reverse transcription reaction was performed for two hours at 42◦C using the following reagents (values in parentheses are final molarity or final concentration): oligo dT primer (Operon, 3.75 μM), dithiothreitol (Invitrogen, 10 mM), First Strand Buffer (Invitrogen, 1×), dNTPs minus dTTP (Invitrogen, 0.5 mM), dTTP (Invitrogen, 50 μM), Cy-labeled dUTP (Perkin-Elmer, 0.625 nM), and Superscript II reverse transcriptase (Invitrogen, 10 U/μL). The reaction was stopped and RNA was hydrolyzed by a 20 minute incubation at 65◦C with NaOH (167 mM) and EDTA (83 mM). After neutralizing by adding HEPES buffer (pH8.0; 294 mM) and sodium acetate (pH5.2; 228 mM), cDNA samples were purified (Gel-Purification Kit, Qiagen, Valencia, CA).
 
 
Hybridization protocol cDNA samples were then dried and resuspended in formamide (56%), sodium citrate buffer (SSC, 3.37×), SDS (1.12%), Denhardts (5.62×), and Polyadenylic acid potassium salt (0.9 mg/ml; Sigma-Aldrich, St. Louis, MO). Samples were boiled for 2 minutes and then applied to microarray slides underneath a LifterSlip (Erie Scientific, Portsmouth, PA). Microarrays were hybridized at 42◦C for 14-18 hours, then washed in a solution of 1.5% SDS and 1X SSC for 5 minutes, a solution of 0.20X SSC for 5 minutes, and two solutions of 0.05X SSC for 10 minutes each.
Scan protocol All arrays were scanned using the GenePix 4100A scanner and GenePix Pro 5.0 software from Axon Instruments (Molecular Diagnostics, Sunnyvale, CA).
Description None
Data processing Visual inspection of the microarray images filtered out florescence most likely not due to labeled cDNA binding; the data from these array elements was discarded. Array elements were only considered for further analysis if at least one channel (Cy3 or Cy5) had greater than 75% of the pixels with intensity values one standard deviation above background levels. All microarray normalization and statistical analyses were performed using the limma package of BioConductor in the program R.
 
Submission date May 01, 2008
Last update date Feb 11, 2009
Contact name Matthew S Lebo
Organization name University of Southern California
Department Molecular and Copmutational Biology
Lab Arbeitman
Street address 1040 Child's Way
City Los Angeles
State/province CA
ZIP/Postal code 90254
Country USA
 
Platform ID GPL6799
Series (2)
GSE11313 Drosophila Gene Expression During Metamorphosis in Wild Type and Germline Minus Pupae
GSE11316 Whole genome microarray analysis of the Drosophila morphogenetic process

Data table header descriptions
ID_REF
VALUE lowess normalized log2 ratio: tud female/ref

Data table
ID_REF VALUE
DAC001A01 null
DAC001A02 null
DAC001A03 -0.523836772
DAC001A04 null
DAC001A05 null
DAC001A06 null
DAC001A07 null
DAC001A08 null
DAC001A09 null
DAC001A10 null
DAC001A11 null
DAC001A12 null
DAC001A13 null
DAC001A14 null
DAC001A15 null
DAC001A16 null
DAC001A17 null
DAC001A18 null
DAC001A19 null
DAC001A20 null

Total number of rows: 15776

Table truncated, full table size 267 Kbytes.




Supplementary file Size Download File type/resource
GSM285838.gpr.gz 1.4 Mb (ftp)(http) GPR
Processed data included within Sample table

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