|
| Status |
Public on Jul 04, 2009 |
| Title |
Musa_DNA_RICE |
| Sample type |
genomic |
| |
|
| Source name |
Musa DNA
|
| Organism |
Musa sp. |
| Characteristics |
tissue: leaf
cultivar: 'Cachac'
|
| Biomaterial provider |
All plant material used was obtained as sterile tissue culture from the Bioversity's International Transit Center (ITC) located in the Laboratory of Tropical Crop Improvement of Division of Crop Biotechnics, Katholieke Universiteit Leuven, Belgium.
|
| Growth protocol |
Plants were grown at a density of 24 plants per table under a standardised light/dark regime of 12/12 h at 26/23oC respectively with a light intensity of around 10,000 lux (300 watts/m2) and a relative humidity of 75%. At the onset of the experiment, all plants were hand-watered until water drained freely from the base of the pots. The control plants were then watered once a day by flooding the tables with a fertigation solution and allowing the pots to stand for 30 min. Water-deficit (drought) conditions were imposed by withholding water supply for in total up to five weeks. Leaf samples consisted of a 5-8 cm wide strip (~3g fresh weight) removed from one side of the middle of the second, fully expanded leaf down from the top of the plant. Samples were snap frozen in liquid nitrogen and stored at -80oC until analysis. All sampling took place between 14:00 and 17:00 pm.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA (gDNA) was isolated from young leaves which had been kept in the dark for 48 h to deplete starch and polysaccharide levels using a modified CTAB method essentially as according to Michiels et al. Contaminating RNA was removed by addition of 2.5 ul of a 10 ug/ml stock solution of RNase and incubation at 37oC for 30 min. To check the quality and quantity of DNA, 1 ul samples of isolated DNA were run on a 1% agarose gel in TAE buffer (0.04 M Tris-acetate; 1 mM EDTA, pH 8) as outlined by Sambrook et al. After staining of the gel with ethidium bromide for 15 min, DNA concentrations were visually estimated by comparison to different amounts of lambda-DNA run at the same time. genomic DNA quality was determined spectrophotometrically using the AU absorbance ratios at 260/280 nm. Samples with a 260/280 ratio of 1.9-2.0 were considered 'pure'.
|
| Label |
biotin
|
| Label protocol |
Musa genomic DNA was labeled using the Bioprime DNA hybridisation System (Invitrogen).
|
| |
|
| Hybridization protocol |
Genomic DNA hybridisation was carried out essentially as described by Hammond et al. In brief, labeled gDNA was hybridised to the Rice Genome Arrays for 16 h at 45oC, using standard Affymetrix hybridisation protocols. This was then followed by the Affymetrix eukaryotic wash protocol that included antibody staining. The GeneChip Genome Arrays were then hybridised with 0.5 ug of target Musa genomic DNA. GeneChip arrays were stained with Streptavidin-Phycoerythrin solution as described by the Affymetrix GeneChip Expression Analysis Technical Manual (http://www.affymetrix.com). Only one genomic DNA hybridisation was performed, as replicate genomic DNA hybridisations all challenge the GeneChip arrays with the whole genome.
|
| Scan protocol |
Scanning done with G2500A GeneArray scanner as described in the Affymetrix GeneChip Expression Analysis Technical Manual.
|
| Description |
Musa DNA hybridized onto Rice array.
|
| Data processing |
From these results a genomic DNA cell intensity file (.cel file) was generated using the Microarray Analysis Suite software (MAS, v5.0, Affymetrix). This .cel file contains the hybridisation intensities between Musa genomic DNA fragments and all the Rice GeneChip oligonucleotide probes. Probes showing a suitably strong cross-hybridisation signal were selected from the .cel file, using a .cel file parser script (Xspecies v 1.1, available with instructions at http://affymetrix.arabidopsis.info/xspecies/) written in the Perl programming language (http://www.perl.com). This Perl script creates a probe mask (.cdf) file, which is compatible with a range of microarray analysis software packages and provides the template for the generation of a signal across the probeset when analyzing the test (Musa) transcriptome (i.e. the RNA .cel files). Specifically it allows information to be extracted from the RNA .cel files for only those probe-pairs whose perfect-match (PM) probe genomic DNA hybridisation intensity value is above a user-defined hybridisation intensity threshold (CDF value). In practice the optimal genomic DNA CDF is determined systematically and empirically using probe masks created with CDF values of between 50 and 1000. A probeset is retained for analysis when it is represented by at least one PM probe-pair per probeset. Therefore one 25 bp probe, identical in sequence to a Musa genomic DNA fragment is the minimum requirement for inclusion of that probeset in the subsequent transcriptome analysis of the target Musa RNA.
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| |
|
| Submission date |
Jun 26, 2009 |
| Last update date |
Jul 03, 2009 |
| Contact name |
Nottingham Arabidopsis Stock Centre (NASC) |
| E-mail(s) |
affy@arabidopsis.info
|
| Phone |
+44 (0)115 951 3237
|
| Fax |
+44 (0)115 951 3297
|
| URL |
http://arabidopsis.info/
|
| Organization name |
Nottingham Arabidopsis Stock Centre (NASC)
|
| Department |
School of Biosciences, University of Nottingham
|
| Street address |
Sutton Bonington Campus
|
| City |
Loughborough |
| ZIP/Postal code |
LE12 5RD |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL2025 |
| Series (1) |
| GSE16865 |
Heterologous microarrays for the study of drought stress in Musa |
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