|
| Status |
Public on Jun 01, 2008 |
| Title |
Yeast mitochondrial associated RNA prep 02 (pu0f2) replicate 2 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
Total RNA extracted from delta puf3 () strain labeled with Cyanine-5 (red).
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
delta puf3 strain
spheroplatic cells
|
| Growth protocol |
Cell grown 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 (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 delta puf3 () strainlabeled with Cyanine-3 (green).
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
delta puf3 strain
Mitochondria extracted from spheroplastic cells
|
| Growth protocol |
Cell grown 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. 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 2 of 2, S. cerevisiae delta puf3 strain grown on galactose medium at 28°C.
|
| 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 |
|
