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Sample GSM239132 Query DataSets for GSM239132
Status Public on Jun 01, 2008
Title Yeast mitochondrial associated RNA with puromycin, prep01 (puro1) replicate 1
Sample type mixed
 
Channel 1
Source name Total RNA extracted from WT (CW252) in presence of 2,1mM puromycin strain labeled with Cyanine-5 (red).
Organism Saccharomyces cerevisiae
Characteristics CW252 WT strain
spheroplatic CW252 cells
Treatment protocol Spheroplastic cells are incubated 10 minutes in presence of 2,1 mM puromycin 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 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 Mitochondrial associated RNA from WT (CW252) strain extracted in presence of 2,1mM puromycin 1M KCllabeled with Cyanine-3 (green).
Organism Saccharomyces cerevisiae
Characteristics CW252 WT strain
Mitochondria extracted from CW252 spheroplastic cells in presence of 2,1mM puromycin 1M KCl.
Treatment protocol Spheroplastic cells are incubated 10 minutes in presence of 2,1 mM puromycin 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 2,1 mM puromycin and 1M KCl. The pellet corresponding to mitochondria is store at -80°C. RNA associated to mitochondria are then extracted using RNEasy Mini Kit (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 1 of 2, S. cerevisiae Wild Type strain grown on galactose medium at 28°C in presence of 2,1 mM puromycin.
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
VALUE -[INV_VALUE]
MEAN_INTENSITY (log2(Cy5) + log2(Cy3)) /2
INV_VALUE log2 (Cy5/Cy3)

Data table
ID_REF VALUE MEAN_INTENSITY INV_VALUE
1 0.115 5.354 -0.115
2 0.115 5.354 -0.115
3 0.023 5.138 -0.023
4 null 5.034 null
5 1.428 8.816 -1.428
6 null 5.038 null
7 -0.096 5.23 0.096
8 null 4.994 null
9 0.213 6.022 -0.213
10 -0.394 7.797 0.394
11 -0.09 9.7 0.09
12 0.253 6.272 -0.253
13 -0.025 5.681 0.025
14 0.077 7.407 -0.077
15 null 5.207 null
16 null 5.014 null
17 -0.788 9.305 0.788
18 0.605 10.121 -0.605
19 null 5.138 null
20 null 5.014 null

Total number of rows: 15743

Table truncated, full table size 362 Kbytes.




Supplementary file Size Download File type/resource
GSM239132.gpr.gz 1.3 Mb (ftp)(http) GPR
GSM239132.tif.gz 12.0 Mb (ftp)(http) TIFF
Processed data included within Sample table

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