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Sample GSM501733 Query DataSets for GSM501733
Status Public on Dec 20, 2010
Title ssdp[31]/ssdp[BG1663] vs. Su(Mrt)[31] e[5] ca/+ biorep 2
Sample type RNA
 
Channel 1
Source name Whole third instar male larvae
Organism Drosophila melanogaster
Characteristics genotype: w[*]/+; ssdp[BG1663]/Su(Mrt)[31] e[5] ca
tissue: Whole third instar male larvae
Growth protocol Larvae were grown in small vials containing colored medium: 7.5g/l agar, 35g/l flour, 50g/l yeast, 55g/l glucose, 2.5ml/l p-Hydroxybenzoic Acid, 4ml/l Propionic Acid and 0.5ml/l Bromophenol. The colored medium allows for more precise staging of the larvae. Towards the end of the third larval stage the larvae cease to feed and the gut clears out. The colored medium can be seen through the live whole larvae. Larvae were collected when the gut was two thirds full and selected or the desired genotype using the GFP marker.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted using Trizol (Life Technologies, Carlsbad, USA), followed by mRNA isolation using an Oligotex poly(A) extraction kit (Qiagen, Valencia, USA).
Label Cy3
Label protocol Purified mRNA (600 ng) was converted to either Cy3- or Cy5-labeled cDNA probes. Each reaction contains 50 mM Tris-HCl pH 8.3, 75 mM KCl, 15 mM MgCl2, 4 mM DTT, 2 mM dNTPs (0.5 mM each), 6 μg Cy3 or Cy5 random 9-mer (Trilink, San Diego, CA), 60 U RNase inhibitor (Ambion, Austin, TX), 600 U MMLV RT RNase (H-) (Promega, Madison, WI) in 75 μl. Labeled Cy3 or Cy5 cDNA products were combined and subsequently de-pooled into three aliquots and purified with ChromaSpin+ TE-30 gel-filtration spin column (Clontech). The probes were then re-pooled, concentrated by ethanol precipitation and resuspended in hybridization buffer.
 
Channel 2
Source name Whole third instar male larvae
Organism Drosophila melanogaster
Characteristics genotype: Su(Mrt)[31] e[5] ca/+
tissue: Whole third instar male larvae
Growth protocol Larvae were grown in small vials containing colored medium: 7.5g/l agar, 35g/l flour, 50g/l yeast, 55g/l glucose, 2.5ml/l p-Hydroxybenzoic Acid, 4ml/l Propionic Acid and 0.5ml/l Bromophenol. The colored medium allows for more precise staging of the larvae. Towards the end of the third larval stage the larvae cease to feed and the gut clears out. The colored medium can be seen through the live whole larvae. Larvae were collected when the gut was two thirds full and selected or the desired genotype using the GFP marker.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted using Trizol (Life Technologies, Carlsbad, USA), followed by mRNA isolation using an Oligotex poly(A) extraction kit (Qiagen, Valencia, USA).
Label Cy5
Label protocol Purified mRNA (600 ng) was converted to either Cy3- or Cy5-labeled cDNA probes. Each reaction contains 50 mM Tris-HCl pH 8.3, 75 mM KCl, 15 mM MgCl2, 4 mM DTT, 2 mM dNTPs (0.5 mM each), 6 μg Cy3 or Cy5 random 9-mer (Trilink, San Diego, CA), 60 U RNase inhibitor (Ambion, Austin, TX), 600 U MMLV RT RNase (H-) (Promega, Madison, WI) in 75 μl. Labeled Cy3 or Cy5 cDNA products were combined and subsequently de-pooled into three aliquots and purified with ChromaSpin+ TE-30 gel-filtration spin column (Clontech). The probes were then re-pooled, concentrated by ethanol precipitation and resuspended in hybridization buffer.
 
 
Hybridization protocol Hybridization was performed in custom-made chambers allowing simultaneous exposure of the probe solution to both slides representing the entire Drosophila transcriptome. Spots (1 μl each) of a 40% suspension (v/v) of 30 μm ceramic microspheres in water were placed at four locations along each side of one slide and allowed to dry. The second slide was placed over the first slide such that the spotted parts of the slides were facing each other and the beads maintained proper spacing between the slides. Hybridization solution was applied at one end and covered the array surfaces by capillary action. Hybridization of labeled cDNA probes was performed in 50 μl 5x SSC, 0.1% SDS, and 1 mM DTT at 60°C for 6 h. The microarrays were washed after hybridization in 1x SSC, 0.1% SDS, 1 mM DTT at 45°C for 10 min, and then in 0.1x SSC, 0.2% SDS, 1 mM DTT at room temperature for 3 min. Microarrays were dried by centrifugation.
Scan protocol Arrays were scanned using an Axon GenePix 3000A fluorescence reader (Molecular Devices Corporation, Union City, USA). GenePix v.4.1 image acquisition software (Molecular Devices Corporation) was used to extract signal for each target element.
Description In the matrix file the data from slide A and B are combined, A is on top.
Data processing The array data was analyzed using R. The raw intensity data normalized within-arrays using the PrintTipLoess algorithm, and next between-arrays, using Quantile. Array elements whose intensity was lower than the median intensity in both channels were discarded. No other background correction method was used. Duplicated elements were not averaged.
 
Submission date Jan 27, 2010
Last update date Dec 20, 2010
Contact name Revital Bronstein
E-mail(s) revitalbronstein@yahoo.com
Organization name Tel-Aviv University
Department Molecular microbiology and biotechnology
Lab Dvelopmental biology
Street address Ramat Aviv
City Tel-Aviv
ZIP/Postal code 69978
Country Israel
 
Platform ID GPL20
Series (1)
GSE20074 Transcription profiling of Drosophila melanogaster SSDP target genes

Data table header descriptions
ID_REF
VALUE PrintTipLoess and Quantile normalized log2 ratio (2 hypomorphic alleles/heterozygote) of gene elements only (minus control elements)

Data table
ID_REF VALUE
10 0.129398228
11 0.119726972
12 0.077544999
13 -0.058527506
14 -0.068418928
15 -0.231959286
16 0.642705394
17 -0.056381001
18 null
19 -0.012091018
20 -0.066126182
21 -0.055026693
22 null
23 -0.186800129
24 null
25 0.035958627
26 0.078073832
27 0.075940291
28 -0.042595896
29 -0.039391481

Total number of rows: 29222

Table truncated, full table size 479 Kbytes.




Supplementary file Size Download File type/resource
GSM501733_B1240486_A.txt.gz 1.3 Mb (ftp)(http) TXT
GSM501733_B1270237_B.txt.gz 1.3 Mb (ftp)(http) TXT
Processed data included within Sample table

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