|
| Status |
Public on Mar 06, 2009 |
| Title |
Genomic DNA - 45 day old leaf sample - mutant f2045 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
45 day old leaf tissue - f2045
|
| Organism |
Oryza sativa Indica Group |
| Characteristics |
tissue: leaf tissue
genotype: IR64 mutant f2045
mutant type: fast neutron
|
| Treatment protocol |
Mutagenesis was induced by treatment of the indica variety IR64 with DEB-treatment, Fast Neutron, or gamma ray (GR) exposure. These mutagenized lines were advanced to M4 or M5 lines prior to DNA extraction by single seed descent
|
| Growth protocol |
A total of 16 mutant populations were grown under greenhouse conditions (standard for rice growth) until 45 days after sowing.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from leaves of 45 day-old greenhouse-grown plants by CTAB extraction and purified by cesium chloride gradient centrifugation. The genomic DNA samples were assayed and quantified by spectrophotometry.
|
| Label |
biotin
|
| Label protocol |
Each sample was biotin labeled using the random priming method with BioPrime® Array CGH Genomic Labeling System (Invitrogen, Carlsbad, CA) following the manufacturer's instruction; a total of 3 µg of genomic DNA from each sample was mixed with 40 µl of 2.5X random primer solutions.
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| |
|
| Channel 2 |
| Source name |
45 day old leaf tissue - wtcheck
|
| Organism |
Oryza sativa Indica Group |
| Characteristics |
tissue: leaf tissue
genotype: wildtype
|
| Growth protocol |
Wildtype samples were grown under greenhouse conditions (standard for rice growth) until 45 days after sowing.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from leaves of 45 day-old greenhouse-grown plants by CTAB extraction and purified by cesium chloride gradient centrifugation. The genomic DNA samples were assayed and quantified by spectrophotometry.
|
| Label |
biotin
|
| Label protocol |
Each sample was biotin labeled using the random priming method with BioPrime® Array CGH Genomic Labeling System (Invitrogen, Carlsbad, CA) following the manufacturer's instruction; a total of 3 µg of genomic DNA from each sample was mixed with 40 µl of 2.5X random primer solutions.
|
| |
|
| |
| Hybridization protocol |
Hybridizations were conducted according to Affymetrix standard protocol for eukaryotic target hybridization. Ten µg of biotinylated fragments were mixed in 200 µl with a final concentration of 0.1 mg/ml sonicated herring sperm DNA in a hybridization buffer with 100 mM 2-N-morpholino-ethane-sulphonic acid (MES), 1 M NaCl, 20 mM EDTA and 0.01% Tween 20, denatured at 99ºC for 5 min and equilibrated at 45ºC for 5 min prior to hybridization. The hybridization mix was then transferred into the Rice GeneChip® cartridge and hybridized at 45ºC for 16 h. The hybridized arrays were washed and stained using EukGE-WS2v5_450 protocol with an Affymetrix GeneChip Fluidics Station 450.
|
| Scan protocol |
The arrays were scanned twice and the intensities averaged with an Affymetrix GeneChip Scanner 3000 using GCOS 1.4.0.036 software (Affymetrix, Santa Clara, CA).
|
| Description |
Genome hybridization using total DNA from f2045
|
| Data processing |
The "affy" package (R/Bioconductor) was used to extract probe level information and examine diagnostics. Perfect match probe data was scale-normalized to the average of the wild type arrays, using a log2 ratio calculation versus wild type at the probe level.
For arrays with replicates, the average probe level intensity was taken prior to data analysis. Similarly, the wild type average probe level intensity was taken prior to data analysis (note: scale normalization occurs AFTER averaging of the intensity for wild type arrays but BEFORE averaging the replicated mutant arrays (G650 and N1856)).
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| |
|
| Submission date |
Mar 03, 2009 |
| Last update date |
Mar 06, 2009 |
| Contact name |
Ramil Paner Mauleon |
| E-mail(s) |
r.mauleon@cgiar.org
|
| Organization name |
International Rice Research Institute
|
| Department |
Crop Research Informatics Laboratory
|
| Lab |
Bioinformatics team
|
| Street address |
IRRI Campus, Pili Drive
|
| City |
Los Banos |
| State/province |
Laguna |
| ZIP/Postal code |
4031 |
| Country |
Philippines |
| |
|
| Platform ID |
GPL2025 |
| Series (1) |
| GSE15071 |
Detection of genomic deletions in rice by genomic DNA hybridization to oligonucleotide microarrays |
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