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Sample GSM271431 Query DataSets for GSM271431
Status Public on May 01, 2008
Title m_pi2_carc / m_tud_carc_GL+
Sample type RNA
 
Channel 1
Source name m_pi2_carc
Organism Drosophila melanogaster
Characteristics Genotype = y[1] w[67c]/+ (pat: pi[[2]]); Sex = M; 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 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 m_tud_carc_GL+
Organism Drosophila melanogaster
Characteristics Genotype = tud[1] bw[1] sp[1] (mat: tud[1] bw[1] sp[1]/CyO); Sex = M; 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 13.377 13.953 0.576 15650 22521
2 767 737
3 829 841
4 1106 1198
5 820 829
6 774 806
7 809 803
8 967 1212
9 838 890
10 9.228 9.208 -0.02 1046 1075
11 9.003 9.014 0.011 927 950
12 9.331 9.272 -0.059 1104 1118
13 9.09 9.02 -0.07 973 956
14 9.629 9.661 0.031 1339 1405
15 9.319 9.297 -0.022 1096 1135
16 10.627 10.687 0.06 2628 2700
17 8.772 8.789 0.017 815 814
18 8.696 8.766 0.071 776 799
19 9.583 9.598 0.015 1296 1353
20 8.901 9.012 0.111 874 944

Total number of rows: 31464

Table truncated, full table size 983 Kbytes.




Supplementary file Size Download File type/resource
GSM271431_109A.gpr.gz 1.2 Mb (ftp)(http) GPR
GSM271431_109B.gpr.gz 1.2 Mb (ftp)(http) GPR
Processed data included within Sample table

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