|
| Status |
Public on Oct 16, 2008 |
| Title |
SB62_Stem_ rep2_at anthesis |
| Sample type |
RNA |
| |
|
| Source name |
stems of SB62 field replicate 2 at anthesis
|
| Organism |
Triticum aestivum |
| Characteristics |
Genotype: SB62 from seri M82 x Babax population, top two internodes at anthesis
|
| Treatment protocol |
The stem tops (peduncle and penultimate internode with leaf sheath attached) were sampled at anthesis between 12:00 and 1:00 pm about 2 days after a heavy rain (the soil in the field was still wet at the time of sampling). Each sample contained 7 to 8 stems from main tillers that were randomly sampled from each plot, immediately dropped into liquid nitrogen, stored at –80oC and used for RNA isolation.
|
| Growth protocol |
8 SB lines were grown under rain-fed conditions with two field replicates of each line in 2005 at the CSIRO Cooper Laboratory at Gatton (latitude 27o 34΄ S, longitude 152o 17΄ E). The site was on a deep fertile prairie loam soil developed on alluvium with a plant available water holding capacity of about 250 mm to a depth of 1.5 m. Plot size in each field trial was 6 m x 1.76 m (8 rows) with an inter-row spacing of 22 cm. Plots were sprayed with herbicides to control weeds and fungicide to prevent foliar diseases.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen fresh stems (peduncle and penultimate internode with leaf sheath attached) were ground to fine powder in liquid nitrogen. Total RNA was isolated from 1 g of wheat stems using Plant RNA Reagent (Invitrogen, California, USA), according to the manufacturer’s instruction. RNA was further purified through a Qiagen RNeasy column (Qiagen, Australia) after pre-treatment with RNase-free DNase I (Xue and Loveridge, 2004 Plant J 37: 326-339).
|
| Label |
Biotin
|
| Label protocol |
Total RNA was quality ascertained using the Agilent Bioanalyser 2100 using the NanoChip protocol. cRNA was prepared from 7ug total RNA using the Affymetrix One Cycle cDNA synthesis kit (Millenium Sciences cat no. 900431). GeneChip Eukaryotic poly-A RNA spikes (Millenium Sciences cat no. 900433) were added to the reaction to determine the labelling efficiency. The subsequent cRNA was cleaned using the Affymetrix GeneChip Sample Cleanup kit (Millenium Sciences cat no. 900371). Upon cleaning of the cRNA the next step was to incorporate biotin into the resultant cRNA using the Affymetrix IVT labelling kit (Millenium Sciences cat no. 900449). After the IVT process the labelled cRNA was cleaned using the above GeneChip Sample Cleanup kit. The quantity of product was ascertained using the Agilent Bioanalyser 2100 using the NanoChip protocol.
|
| |
|
| Hybridization protocol |
A total of 20ug of biotinylated cRNA was fragmented to the 50-200bp size range and quality control checked using the Bioanalyser 2100 using the NanoChip protocol. Samples that pass this checkpoint are then prepared for hybridisation to the Affymetrix Wheat Genome GeneChip by preparing a probe cocktail (cRNA at 0.05ug/ul) that includes 1x Hybridisation Buffer (100mM MES, 1m NaCl, 20mM EDTA, 0.01% Tween-20), 0.1mg/ml Herring Sperm DNA, 0.5mg/ml BSA, and 7% DMSO. A total hybridisation volume of 300ul is prepared for each sample and 200ul loaded into a Wheat Genome GeneChip. The chip is hybridised at 45oC for 16 hours in an oven with a rotating wheel at 60rpm. After hybridisation the chip is washed and stained with SAPE using the appropriate fluidics script in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
The chips are scanned using the Affymetrix GeneChip Scanner 3000
|
| Description |
peduncle and penultimate internodes with leaf sheath attached, at anthesis
|
| Data processing |
The scanner operating software, GCOS, converts the signal on the chip into a DAT file, which will then be used for generating subsequent CEL files for analysis. The raw data in the Affymetrix CEL files of all 16 arrays representing two biological replicates of each of 8 genotypes were normalized using robust multiarray average (Irizarry et al. 2003, Biostatistics 4:249-264), using the default settings for the Affymetrix package within Bioconductor, running within the R statistical programming environment (http://www.r-project.org/).
|
| |
|
| Submission date |
Dec 04, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Gang-Ping Xue |
| E-mail(s) |
Gang-Ping.Xue@csiro.au
|
| Phone |
61 7 3214 2354
|
| Fax |
61 7 3214 2920
|
| Organization name |
CSIRO
|
| Department |
Plant Industry
|
| Street address |
306 Carmody Rd., St Lucia
|
| City |
Brisbane |
| State/province |
Qld |
| ZIP/Postal code |
4067 |
| Country |
Australia |
| |
|
| Platform ID |
GPL3802 |
| Series (1) |
| GSE9767 |
Genotypic differences in water soluble carbohydrate metabolism in stem |
|
