|
| Status |
Public on Apr 02, 2011 |
| Title |
Pooled rpfF2 |
| Sample type |
RNA |
| |
|
| Source name |
RNA isolated from three independent cultures of rpfF mutant
|
| Organism |
Xanthomonas oryzae pv. oryzae |
| Characteristics |
isolate: Indian
genotype: rpfF1 mutant
growth time: 28 hours
|
| Growth protocol |
Xanthomonas oryzae pv. oryzae wild type and DSF-deficient rpfF mutant cells were grown in Peptone sucrose medium, 200 rpm, 28o C and inoculated in the secondary culture at a cell density of 10^5 cells/ml (inoculum density) and grown for 28 hours under similar conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol following the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Poly (A)-tails were added to the 3’-end of RNA by using the A-plus Poly (A) polymerase tailing kit (Epicentre Biotechnologies). The samples were labeled using Agilent Quick Amp Kit PLUS (Part number: 5190-0442). Five hundred nanograms each of the samples were incubated with reverse trancription mix at 42°C and converted to double-stranded cDNA primed by oligodT with a T7 polymerase promoter. The cleaned up double-stranded cDNA were used as template for aRNA generation. cRNA was generated by in vitro transcription and the dye Cy3 CTP(Agilent) was incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried out at 40°C. Labeled cRNA was cleaned up and quality assessed for yields and specific activity.
|
| |
|
| Hybridization protocol |
The labeled cRNA samples were hybridized onto a Genotypic designed Custom Gene Expression Xanthomonas oryzae 8x15k (AMADID: 25096). 600 ng of Cy3-labeled samples were fragmented and hybridized. Fragmentation of labeled cRNA and hybridization were done using the Gene Expression Hybridization kit of Agilent (Part Number 5188-5242). Hybridization was carried out in Agilent’s Surehyb Chambers at 65º C for 16 hours. The hybridized slides were washed using Agilent Gene Expression wash buffers (Part No: 5188-5327).
|
| Scan protocol |
Scanned on an Agilent Technologies G2505C scanner. Images were quantified using Agilent Feature Extraction Software 10.5.1.1.
|
| Description |
Gene expression after 28 hours of growth in Peptone sucrose media.
|
| Data processing |
The normalization was done using GeneSpring GX 11 Software.
|
| |
|
| Submission date |
Mar 07, 2011 |
| Last update date |
Apr 02, 2011 |
| Contact name |
Genotypic technology |
| E-mail(s) |
sudha.rao@genotypic.co.in
|
| Organization name |
Genotypic Technology
|
| Street address |
259, Apoorva 4th cross,80 feet Road,RMV 2ND STAGE
|
| City |
Bangalore |
| State/province |
Karnataka |
| ZIP/Postal code |
560094 |
| Country |
India |
| |
|
| Platform ID |
GPL13231 |
| Series (1) |
| GSE27809 |
Xanthomonas oryzae pv. oryzae (Indian Isolate): Wild type vs. rpfF mutant |
|
