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Sample GSM271437 Query DataSets for GSM271437
Status Public on May 01, 2008
Title f_tudCy_carc_GL+ / f_pi2_carc
Sample type RNA
 
Channel 1
Source name f_tudCy_carc_GL+
Organism Drosophila melanogaster
Characteristics Genotype = tud[1] bw[1] sp[1]/CyO DTS (mat: tud[1] bw[1] sp[1]/CyO DTS); Sex = F; Stage = Adult; Tissue = Carcass
Growth protocol Flies were grown at 22C on standard cornmeal medium (Tucson, AZ) for 3 to 5 days post eclosion and mated. Adult flies were then collected and quick frozen on dry ice and total RNA was extracted (Andrews et al, Genome Research 10:2030-2043) using Trizol reagent (GibcoBRL, Gaithersburg, MD).
Extracted molecule total RNA
Extraction protocol Adult flies collected and quick frozen on dry ice and total RNA was extracted (Andrews et al, Genome Research 10:2030-2043) using Trizol reagent (GibcoBRL, Gaithersburg, MD).
Label Cy3
Label protocol Probes were labeled with Cy3 (Probe 1) or Cy5 (Probe 2). To synthesize probes, mRNA was isolated by a single round of poly(A) selection using Oligotex resin (Qiagen, Valencia, CA). The purified mRNA was quantified using RiboGreen dye (Molecular Probes) in a fluorescent assay. RiboGreen dye was diluted 1:200 (v/v final) and mixed with known RNA concentrations (determined by absorbance at 260 nm) ranging from 1 to 5000 ng/ml. A Millennium RNA size ladder (Ambion, Austin, TX) was used to generate standard curves and unknown samples were diluted as necessary. Fluorescence was measured in 96-well plates with a FLUOstar fluorometer (BMG Lab Technologies, Germany) fitted with 485 nm (excitation) and 520 nm (emission) filters. Between 25 and 100 ng mRNA were separated on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), to examine the mRNA size distribution. 200 ng of purified mRNA were converted to either a Cy3- or Cy5-labeled cDNA probe using a custom labeling kit (Incyte Genomics). Each reaction contained 50 mM Tris-HCl pH 8.3, 75 mM KCl, 15 mM MgCl2, 4 mM DTT, 2 mM dNTPs (0.5 mM each), 2 µg Cy3 or Cy5 random 9mer (Trilink, San Diego, CA), 20 U RNase inhibitor (Ambion), 200 U MMLV RNase H-free reverse transcriptase (Promega, Madison, WI) and mRNA. Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on a size exclusion column, concentrated by ethanol precipitation and resuspended in hybridization buffer.
 
Channel 2
Source name f_pi2_carc
Organism Drosophila melanogaster
Characteristics Genotype = y[1] w[67c]/+ (pat: pi[[2]]); Sex = F; Stage = Adult; Tissue = Carcass
Growth protocol Flies were grown at 29C on standard cornmeal medium (Tucson, AZ) until the first instar stage, and then transferred to 22C until 3 to 5 days post eclosion and mated. Adult flies were then collected and quick frozen on dry ice and total RNA was extracted (Andrews et al, Genome Research 10:2030-2043) using Trizol reagent (GibcoBRL, Gaithersburg, MD).
Extracted molecule total RNA
Extraction protocol Adult flies collected and quick frozen on dry ice and total RNA was extracted (Andrews et al, Genome Research 10:2030-2043) using Trizol reagent (GibcoBRL, Gaithersburg, MD).
Label Cy5
Label protocol Probes were labeled with Cy3 (Probe 1) or Cy5 (Probe 2). To synthesize probes, mRNA was isolated by a single round of poly(A) selection using Oligotex resin (Qiagen, Valencia, CA). The purified mRNA was quantified using RiboGreen dye (Molecular Probes) in a fluorescent assay. RiboGreen dye was diluted 1:200 (v/v final) and mixed with known RNA concentrations (determined by absorbance at 260 nm) ranging from 1 to 5000 ng/ml. A Millennium RNA size ladder (Ambion, Austin, TX) was used to generate standard curves and unknown samples were diluted as necessary. Fluorescence was measured in 96-well plates with a FLUOstar fluorometer (BMG Lab Technologies, Germany) fitted with 485 nm (excitation) and 520 nm (emission) filters. Between 25 and 100 ng mRNA were separated on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), to examine the mRNA size distribution. 200 ng of purified mRNA were converted to either a Cy3- or Cy5-labeled cDNA probe using a custom labeling kit (Incyte Genomics). Each reaction contained 50 mM Tris-HCl pH 8.3, 75 mM KCl, 15 mM MgCl2, 4 mM DTT, 2 mM dNTPs (0.5 mM each), 2 µg Cy3 or Cy5 random 9mer (Trilink, San Diego, CA), 20 U RNase inhibitor (Ambion), 200 U MMLV RNase H-free reverse transcriptase (Promega, Madison, WI) and mRNA. Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on a size exclusion column, concentrated by ethanol precipitation and resuspended in hybridization buffer.
 
 
Hybridization protocol Probes were added between two subarray slides and developed as described (Yue et al.2001, NAR. 29:e41).
Scan protocol Microarrays were scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially captured using GenePix Pro 4.1 (Axon Instruments, Foster City CA).
Description Expression is assayed in vairous adult tissues with germline ablated directly or genetically. Additional descriptive information is available at Parisi et.al (2004) Genome Biol. 2004;5(6):R40. PMID: 15186491
Data processing Normalization by within-slide print tip loess, along with subsequent analyses, were performed using the bioconductor package LIMMA 2.12.0 (Smyth et.al, 2004).
 
Submission date Mar 06, 2008
Last update date Apr 25, 2012
Contact name Brian Oliver
E-mail(s) briano@nih.gov
Phone 301-204-9463
Organization name NIDDK, NIH
Department LBG
Lab Developmental Genomics
Street address 50 South Drive
City Bethesda
State/province MD
ZIP/Postal code 20892
Country USA
 
Platform ID GPL20
Series (1)
GSE11017 Contribution of germline to sex-biased expression in Drosophila melanogaster

Data table header descriptions
ID_REF ID to link data back to GPL20 platform
Log2Cy3 Log2 transformed intensity signal from Cy3 channel. Values are normalized within each array by print tip Loess and across the series using quantile normalization in Bioconductor.
Log2Cy5 Log2 transformed intensity signal from Cy5 channel. Values are normalized within each array by print tip Loess and across the series using quantile normalization in Bioconductor.
VALUE Log2 transformed ratio of corrected Cy5/Cy3 signal calculated by taking the corrected Log2Cy5 signal value and subtracting the Log2Cy3 signal value for each element
Cy3_SIGNAL Raw median signal intensity data from from Cy3 channel acquired by Genepix
Cy5_SIGNAL Raw median signal intensity data from from Cy5 channel acquired by Genepix

Data table
ID_REF Log2Cy3 Log2Cy5 VALUE Cy3_SIGNAL Cy5_SIGNAL
1 13.525 14.409 0.885 15771 41789
2 551 688
3 576 658
4 838 1122
5 570 567
6 544 568
7 575 821
8 677 951
9 602 725
10 9.332 9.446 0.113 864 1341
11 8.889 8.933 0.044 688 889
12 9.207 9.26 0.053 809 1174
13 9.01 9.029 0.019 733 974
14 9.877 9.801 -0.075 1202 1782
15 9.484 9.527 0.044 942 1427
16 10.67 10.693 0.022 2137 3457
17 8.561 8.556 -0.005 579 592
18 8.403 8.541 0.139 516 560
19 9.76 9.809 0.048 1117 1787
20 8.908 8.909 0 698 873

Total number of rows: 31464

Table truncated, full table size 978 Kbytes.




Supplementary file Size Download File type/resource
GSM271437_B1230495_A.txt.gz 1.3 Mb (ftp)(http) TXT
GSM271437_B1270241_B.txt.gz 1.3 Mb (ftp)(http) TXT
Processed data included within Sample table

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