|
| Status |
Public on Jun 30, 2010 |
| Title |
Mycelium 96h replicate 46 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
M96h
|
| Organism |
Podospora anserina |
| Characteristics |
growth condition: harvested after 96 hours of growth in darkness, on M2 media covered with a cheethcloth
mating type: mat+
|
| Growth protocol |
P. anserina strains used in this study are the wild type S and s strains of either mat+ or mat- mating type. Growth was carried out on a minimal synthetic medium at 27°C (Esser K.,1974; pp. 531-51 in Handbook of Genetics)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Strains were grown on cellophane sheet (cat#1650193, BioRad, Marne la Coquette, France) or cheesecloth (Sefar Nitex 03-48/31, Dominique Dutscher, Brumath, France) placed on Petri-dishes. In order to collect sufficient amounts of biological materials (20 to 100 mg), 1 to 5 Petri-dishes were simultaneously inoculated. Biological material was scraped from cellophane sheets or cheesecloth and flash frozen with buffer RLT (Qiagen, Courtaboeuf, France) in liquid nitrogen. The biological material was grinded with a Mikro-Dismembrator (Sartorius, Aubagne, France) in nitrogen freezed vessels. After removing cell debris by centrifugation, nucleic acids were passed through a Qiashreddrer (Qiagen) for sharing DNA and total RNA was purified on RNeasy Plant Mini Kit columns (Qiagen), with an additional DNase treatment. The quality and quantity of the total RNA was determined by using a NanoDrop ND-1000 spectrophotometer and the Bionalyzer 2100 system (Agilent).
|
| Label |
Cy3
|
| Label protocol |
Labelling experiments were done following the two-color microarray-based gene expression analysis instructions (version 5.0, February 2007) as described by the manufacturer (Agilent). One microgram aliquots of total RNA were amplified and Cy-labelled with the Agilent’s Low RNA input fluorescent linear amplification (LRILAK) PLUS kit (Agilent) along with Agilent's Two-Color RNA Spike-in Kit. The labelling efficiency and the product integrity were checked by using a NanoDrop ND-1000 spectrophotometer and the Bionalyzer 2100 system (Agilent)
|
| |
|
| Channel 2 |
| Source name |
Common reference (pool of four conditions M48h, M96h, C48h and C96h)
|
| Organism |
Podospora anserina |
| Characteristics |
biosource 1: M48h
biosource 1 growth condition: mat+ and mat- S strain harvested after 48 hours of growth on M2 media covered with a sheet of cellophane.
biosource 2: M96h
biosource 2 growth condition: mat+ and mat- S strain harvested after 96 hours of growth on M2 media covered with a sheet of cellophane.
biosource 3: C48h
biosource 3 growth condition: mat+ and mat- S strain cultivated for 96 hours on M2 media covered with a cheesecloth, spermatization with mat- and mat+ (respectively) S spermatia and harvesting after 48 h.
biosource 4: C96h
biosource 4 growth condition: mat+ and mat- S strain cultivated for 96 hours on M2 media covered with a cheesecloth, spermatization with mat- and mat+ (respectively) S spermatia and harvesting after 96 h.
|
| Growth protocol |
P. anserina strains used in this study are the wild type S and s strains of either mat+ or mat- mating type. Growth was carried out on a minimal synthetic medium at 27°C (Esser K.,1974; pp. 531-51 in Handbook of Genetics)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Strains were grown on cellophane sheet (cat#1650193, BioRad, Marne la Coquette, France) or cheesecloth (Sefar Nitex 03-48/31, Dominique Dutscher, Brumath, France) placed on Petri-dishes. In order to collect sufficient amounts of biological materials (20 to 100 mg), 1 to 5 Petri-dishes were simultaneously inoculated. Biological material was scraped from cellophane sheets or cheesecloth and flash frozen with buffer RLT (Qiagen, Courtaboeuf, France) in liquid nitrogen. The biological material was grinded with a Mikro-Dismembrator (Sartorius, Aubagne, France) in nitrogen freezed vessels. After removing cell debris by centrifugation, nucleic acids were passed through a Qiashreddrer (Qiagen) for sharing DNA and total RNA was purified on RNeasy Plant Mini Kit columns (Qiagen), with an additional DNase treatment. The quality and quantity of the total RNA was determined by using a NanoDrop ND-1000 spectrophotometer and the Bionalyzer 2100 system (Agilent).
|
| Label |
Cy5
|
| Label protocol |
Labelling experiments were done following the two-color microarray-based gene expression analysis instructions (version 5.0, February 2007) as described by the manufacturer (Agilent). One microgram aliquots of total RNA were amplified and Cy-labelled with the Agilent’s Low RNA input fluorescent linear amplification (LRILAK) PLUS kit (Agilent) along with Agilent's Two-Color RNA Spike-in Kit. The labelling efficiency and the product integrity were checked by using a NanoDrop ND-1000 spectrophotometer and the Bionalyzer 2100 system (Agilent)
|
| |
|
| |
| Hybridization protocol |
Hybridization experiments were done following the two-color microarray-based gene expression analysis instructions (version 5.0, February 2007) as described by the manufacturer (Agilent). 825 ng of each of the Cy3- and Cy5-labeled targets were mixed and incubated on an Agilent microarray slide for 17 hours at 65°C, in a rotating oven at 10 rpm for a 4x44K array format, using an Agilent in situ hybridization kit. The slides were washed in Agilent’s Gene Expression Wash Buffers following manufacturer’s instructions, then, any traces of solution were removed by centrifugation at 800 rpm for 1 min.
|
| Scan protocol |
Microarrays were scanned using the Agilent DNA microarray Scanner model G2567AA at 5 microns resolution using the extended dynamic range (XDR) feature.
|
| Description |
Biological replicate 4 of 4. Mycelium after 96h of growth
|
| Data processing |
Spot and background intensities were extracted with the Feature Extraction (FE, v9.5.3) software (Agilent) using the GE2-v4_95_Feb07 default protocol. Preliminary array quality was assessed through the use of Agilent control features as well as spike-in controls (Agilent 2-Color Spike-in Kit for RNA experiment). Subsequent flagging was done according to the GenePix Pro software (Molecular Devices Syunnyvale, CA, USA) nomenclature, including four levels of flags (good (100), bad (-100), not found (-50), moderate (0)). FE software raw data from arrays issued of GPL10115 platform were processed with the MAnGO software with no background subtraction and the Printtip loess normalization method (Smyth G.K. and Speed T., 2003, Methods). FE software normalized data (Local background was subtracted and LOWESS normalization) from arrays issued of GPL10116 platform were processed with the MAnGO. Moderate t-test with adjustment of p-values was computed to measure the significance with each expression difference.
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| |
|
| Submission date |
Mar 10, 2010 |
| Last update date |
Jun 30, 2010 |
| Contact name |
Frederique Bidard |
| E-mail(s) |
frederique.bidard-michelot@ifpen.fr
|
| Organization name |
Institut de Génétique et Microbiologie UMR 8621
|
| Department |
SIGNAL TRANSDUCTION, REPRODUCTION & DEGENERATIVE PROCESSES
|
| Street address |
Bâtiment 400
|
| City |
Orsay |
| ZIP/Postal code |
91405 |
| Country |
France |
| |
|
| Platform ID |
GPL10115 |
| Series (1) |
| GSE20734 |
A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina |
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