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Sample GSM238956 Query DataSets for GSM238956
Status Public on Jan 28, 2008
Title mnn++ 4-6 hours / cntrl1 4-6 hours - 76
Sample type RNA
 
Channel 1
Source name mnn++ 4-6 hours
Organism Drosophila melanogaster
Characteristics mnn overexpression, 4-6 hours old embryos
Growth protocol Flies were grown at 25°C on PB medium (KD Medical, Columbia, MD) for 3 to 5 days post eclosion and mated. Embryos 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 Embryos 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 cntrl1 4-6 hours
Organism Drosophila melanogaster
Characteristics control sample, 4-6 hour old embryos
Growth protocol Flies were grown at 25°C on PB medium (KD Medical, Columbia, MD) for 3 to 5 days post eclosion and mated. Embryos 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 Embryos 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 Menin overexpression mutants were generated using transgenic lines bearing the full-length Mnn1 cDNA (Guru et al., 2001) under the control of the yeast GAL4 UAS promoter (Brand and Perrimon, 1993). The constructs were introduced into flies by P-element mediated transgenesis (Rubin and Spradling, 1982). Mutants are described in Cerrato et al., Dev Biol. 2006 Oct 1;298(1):59-70. Control samples are flies that have the same driver, but do not have the without the Mnn1 overexpressing P-element construct.
Data processing Normalization by within-slide print tip loess and between-slide quantile, along with subsequent analyses, were performed using the bioconductor package LIMMA 2.12.0 (Smyth et.al, 2004).
 
Submission date Oct 19, 2007
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)
GSE9425 Expression profile analysis of menin1 mutants

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 14.369 14.257 -0.112 28589 13586
2 9211 3485
3 6323 2566
4 20228 9063
5 21878 17234
6 21878 17234
7 15501 13744
8 16282 12630
9 6987 6415
10 11.266 11.341 0.074 2967 2371
11 7.393 7.441 0.048 186 160
12 7.211 7.371 0.16 163 152
13 9.162 9.2 0.038 761 617
14 7.564 7.492 -0.072 211 166
15 8.725 8.847 0.121 481 452
16 8.48 8.603 0.123 397 362
17 9.345 9.512 0.167 860 749
18 6.978 6.994 0.016 137 113
19 6.348 6.161 -0.187 83 66
20 8.505 8.465 -0.04 402 328

Total number of rows: 31464

Table truncated, full table size 954 Kbytes.




Supplementary file Size Download File type/resource
GSM238956_a.gpr.gz 236.8 Kb (ftp)(http) GPR
GSM238956_b.gpr.gz 232.3 Kb (ftp)(http) GPR
Processed data included within Sample table

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