|
| Status |
Public on Jan 01, 2009 |
| Title |
Wheat var. Kennedy_control_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Crown tissue of Wheat variety Kennedy, mock inoculated, 24hr
|
| Organism |
Triticum aestivum |
| Characteristics |
Fusarium pseudograminearum isolate CS3096 or water was used to inoculate the crown tissue of the wheat (Triticum aestivum) cultivar Kennedy. RNA was extracted from 2cm of crown tissue, 24hr after mock or fungal inoculation.
|
| Biomaterial provider |
CSIRO
|
| Treatment protocol |
Two week old wheat plants were inoculated by placing the pots horizontally and applying 10uL of a 10^6 spores/ml spore suspension of F. pseudograminearum or water to the leaf sheaf approximately 2cm from the soil surface. The plants were kept horizontal at saturating humidity using perspex hoods for 24hrs before the crown tissue was harvested.
|
| Growth protocol |
Plants were grown in a controlled environment growth room under 500umol/m/s PAR with a 7hr photoperiod. Day/night conditions were 25C/15C and 65/95% relative humidity, respectively. Plants were grown in small pots, approximately 5cm, filled with sterile soil containing two seeds sown below the surface. Plants were watered every second day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the caesium chloride extraction method described by Chirgwin et al., 1979. RNA extraction was followed by a DNase treatment using a RNase-free DNase kit (QIAGEN) and column purified using a MiniElute kit (QIAGEN) according to manufacturer's instructions. Samples were dissolved in nuclease free water and sent to the Australian Genome Research Facility for cDNA synthesis, labelling, hybridisation to the Affymetrix GeneChip Wheat Genome Array, washing and scanning.
|
| Label |
biotin
|
| Label protocol |
Refer to the AGRF website http://www.agrf.org.au for details of the protocol used.
|
| |
|
| Hybridization protocol |
Refer to the AGRF website http://www.agrf.org.au for details of the protocol used.
|
| Scan protocol |
Refer to the AGRF website http://www.agrf.org.au for details of the protocol used.
|
| Description |
n/a
|
| Data processing |
GeneSpring 7.3 (Agilent Technologies) was used for data analysis. Each CHP file was imported into GeneSpring running the Wheat GeneChip Genome and then associated with its matching CEL file. A final normalised ratio was obtained by applying an additional “per probe set” normalisation. This was achieved by dividing the raw value for each probe set by the median value for that probe set in the whole experiment. This centred the data near a value of 1 and ensured ease of identification of differentially expressed probe sets. Statistical analysis was performed to identify differentially expressed genes in Fp inoculated samples compared to mock inoculated samples using ANOVA with a p<0.05 across all replicates. Induction or repression of differentially expressed genes was determined by dividing each replicate raw signal value from Fp inoculated tissue by the average of the raw signal from the mock inoculated tissue. Genes were classes as induced or repressed if their change in expression was greater than 1.5 fold.
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| |
|
| Submission date |
Nov 18, 2008 |
| Last update date |
Nov 21, 2008 |
| Contact name |
Brendan Kidd |
| E-mail(s) |
brendan.kidd@csiro.au
|
| Organization name |
CSIRO
|
| Department |
Plant Industry
|
| Street address |
306 Carmody Road
|
| City |
St Lucia |
| State/province |
QLD |
| ZIP/Postal code |
4067 |
| Country |
Australia |
| |
|
| Platform ID |
GPL3802 |
| Series (1) |
| GSE13660 |
Gene expression analysis of the wheat response to infection by Fusarium pseudograminearum |
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