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Sample GSM239131 Query DataSets for GSM239131
Status Public on Jun 01, 2008
Title Yeast mitochondrial associated RNA with cycloheximide, prep02 (puro2) Dye swap replicate 2
Sample type mixed
 
Channel 1
Source name Mitochondrial associated RNA from WT (CW252) strain extracted in presence of 200µg/ml cycloheximidelabeled with Cyanine-5 (red).
Organism Saccharomyces cerevisiae
Characteristics CW252 WT strain
Mitochondria extracted from CW252 spheroplastic cells
Treatment protocol Spheroplastic cells are incubated 10 minutes in presence of 200µg/ml cycloheximide before being harvested
Growth protocol Cell growth overnight at 28°C in galactose rich medium (1% bactopeptone, 1% yeast extract, 2% galactose, 0.1% KH2PO4 and 0.12% (NH4)2SO4) to an OD = 1.0
Extracted molecule other
Extraction protocol Spheroplastic cell are fractionated by differential centrifugation in presence of 200µg/ml cycloheximide. The pellet corresponding to mitochondria is store at -80°C. RNA associated to mitochondria are then extracted using RNEasy Mini Kit (Quiagen)
Label Cy5
Label protocol 1.5 µg RNA are retro transcribed using Superscript III retro transcriptase. The cDNA are purified using QiaQuick column (Quiagen) and incubated 1 hour at room temperature in presence of 0,05M NaBicarbonate and NHS-ester Cy5.The coupling is stoped by addition of hydroxylamine to a final concentration of 0,6M. Labeled cDNA are purified using QiaQuick column.
 
Channel 2
Source name Total RNA extracted from WT (CW252) in presence of 200µg/ml cycloheximide strain labeled with with Cyanine-3 (green).
Organism Saccharomyces cerevisiae
Characteristics CW252 WT strain
Total RNAs
Treatment protocol Spheroplastic cells are incubated 10 minutes in presence of 200µg/ml cycloheximide before being harvested
Growth protocol Cell growth overnight at 28°C in galactose rich medium (1% bactopeptone, 1% yeast extract, 2% galactose, 0.1% KH2PO4 and 0.12% (NH4)2SO4) to an OD = 1.0
Extracted molecule total RNA
Extraction protocol Hot phenol extract procedure followed by RNEasy Mini Kit purification step (Quiagen)
Label Cy3
Label protocol 1.5 µg RNA are retro transcribed using Superscript III retro transcriptase. The cDNA are purified using QiaQuick column (Quiagen) and incubated 1 hour at room temperature in presence of 0,05M NaBicarbonate and NHS-ester Cy3.The coupling is stoped by addition of hydroxylamine to a final concentration of 0,6M. Labeled cDNA are purified using QiaQuick column.
 
 
Hybridization protocol 1.5µg of labelled cDNA is mixed with one volume of 2X GE buffer (Agilent) and 10% of blocking agent. The hybridization sample volume is dispense on the selected microarray and incubated overnight at 65¡C in a hybridization oven (Agilent). The array slides are washed 1 minute in "wash 1 buffer" at room temperature and one minute in "wash 2 buffer" at 37¡C. The array slides are then air dryed and ready for scaning.
Scan protocol Scanned on a genepix 4000B scanner (Axon)
Description Biological replicate 2 of 2, S. cerevisiae Wild Type strain grown on galactose medium at 28°C in presence of 200µg/ml cycloheximide.
Data processing Scanning software: GenePix Pro 6.1
Normalization using Goulphar WebTool http://transcriptome.ens.fr/goulphar/
Genepix flags are removed from the analysis. Spots are considered as saturating spot above 50000 and removed. Background is not subtracted from the main signal. Global Lowess normalisation method.

VALUE column reports log2(Mitochondrial associated RNA/Total RNA) ratios.
 
Submission date Oct 22, 2007
Last update date Aug 14, 2011
Contact name Stéphane LE CROM
Organization name École normale supérieure
Department Biology Institute - IBENS
Lab Genomic platform
Street address 46 rue d'Ulm
City Paris
ZIP/Postal code 75013
Country France
 
Platform ID GPL6035
Series (1)
GSE9393 Looking for mitochondrial associated RNA

Data table header descriptions
ID_REF ENS feature number
R_MEDIAN_SIGNAL Mean foreground intensity Cy5
G_MEDIAN_SIGNAL Mean foreground intensity Cy3
VALUE log2 (Cy5/Cy3)
MEAN_INTENSITY (log2(Cy5) + log2(Cy3)) /2

Data table
ID_REF R_MEDIAN_SIGNAL G_MEDIAN_SIGNAL VALUE MEAN_INTENSITY
1 32 32 0.062 5
2 29 31 -0.098 4.906
3 null null null null
4 null null null null
5 294 483 0.802 8.558
6 null null null null
7 28 30 -0.135 4.857
8 null null null null
9 54 135 -0.301 6.416
10 217 180 1.794 7.627
11 217 1012 -0.745 8.872
12 30 31 -0.033 4.931
13 null null null null
14 251 230 1.674 7.909
15 null null null null
16 null null null null
17 747 419 2.255 9.128
18 384 2859 -1.573 10.033
19 null null null null
20 null null null null

Total number of rows: 15744

Table truncated, full table size 394 Kbytes.




Supplementary file Size Download File type/resource
GSM239131.gpr.gz 1.3 Mb (ftp)(http) GPR
GSM239131.tif.gz 10.9 Mb (ftp)(http) TIFF
Processed data included within Sample table

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