Affymetrix GeneChip Wheat Genome Array [wheat]

Co-ordinated expression of amino acid metabolism in response to N and S deficiency during wheat grain filling

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Transcripomic analysis of leaf gene expression in S and N-deficient winter wheat during grain development. Tissue was harvested at anthesis and 7, 14 and 21 days post anthesis from experimental field plots.

GrowthCondition

Transcription profiling time series of leaves from winter wheat grown under S and N-deficient conditions

Transcripomic analysis of leaf gene expression in S and N-deficient winter wheat during grain development. Tissue was harvested at anthesis and 7, 14 and 21 days post anthesis from experimental field plots.


Affymetrix:Protocol:Hybridization-Unknown

P-AFFY-2

Labeling of samples was carried out according to the manufacturer’s standard protocol

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-126541

Farm-scale outdoor field trial planted at Rothamsted Research annually. Winter wheat (current variety Hereward (RAGT Seeds Ltd, Cambridge, UK)) is grown under a range of agronomic regimes. For details see Poutlon et al 2005.  For this experiment wheat plants were harvested from N-deficient (N1), S-deficient (-S) and control fertiliser (N4) fed plots. Seed were drilled in mid October at a rate of 350 seed/m2.  Fertlisers were applied as follows at the end of April:-  N1, 48kg/ha ammonium nitrate, 90kg/ha potassium sulfate N4, 192kg/ha ammonium nitrate, 90kg/ha potassium sulfate -S, 192kg/ha ammonium nitrate, 90kg/ha potassium chloride  Grain and leaf material for array experiments was harvested weekly from anthesis.  The open air nature of the field trial meant ambient temperature prevailed throughout the experiment.   References: Poulton PR (1995) The importance of long-term trials in understanding sustainable farming systems: the Rothamsted experience. Australian Journal of Experimental Agriculture 35: 825-834

P-MEXP-126866

Raw CEL file data was preprocessed using GC-RMA algorithm (Wu et al. 2004)  Values less than 0.1 were transformed to 0.1  Per Gene normalisation was then carried out by dividing all data values by the median value across all experimental samples and replicates.  References: Wu ZJ, Irizarry RA, Gentleman R, Martinez-Murillo F, Spencer F (2004) A model-based background adjustment for oligonucleotide expression arrays. Journal of the American Statistical Association 99: 909-917

P-MEXP-89691

Total RNA was isolated from A. thaliana tissue using the method of Verwoerd et al. (1989). Tissue was ground to a very fine powder in liquid nitrogen using a pestle and mortar and 100-200 mg (aproximately 500 ul of powder) was transferred to a liquid N chilled 2ml microfuge tube. 1000 ul of extraction buffer (0.1 M LiCl, 0.1 M Tris-HCl pH 8.0, 10 mM EDTA, 0.1% (w/v) SDS mixed 1:1 with phenol on day of use) preheated to 85¢XC was added and the tube vortexed for 20 seconds. 500 ul of chloroform/IAA (24:1 ratio) was then added to the tube before vortexing for a further 30 seconds. Keep on ice until all tubes are processed. The tubes were centrifuged at 10000 xg for 10 mins in a bench-top microfuge at 4¢XC and the aqueous phase transferred to a clean tube. To minimise the risk of RNA hydrolysis by contaminating RNases all the following steps were carried out at 4¢XC, using plasticware which had been autoclaved at 121¢XC for 25 mins and touched only with gloved hands. RNA was precipitated by adding an equal volume of 4 M LiCl, mixing and incubating over night at 4¢XC. Next day tubes were centrifuged at 10000 xg for 15 mins, the supernatant removed and the pellet allowed to air dry before resuspending in 250 ƒÝl sterile distilled water treated with DEPC (mixing distilled water with 0.1% DEPC before autoclaving inactivates RNases). RNA was then precipitated by 2 vol EthOH, 0.1 vol 3M NaAcetate for 30 min  at ¡V80degC, spun down as before and washed in 70% EthOH 30% DEPC H2O. The pellet was disolved in 50ul DEPC H2O. RNA was then cleaned using Qiagen RNeasy columns following manufacturers protocol.   Verwoerd TC, Dekker BMM and Hoekema A (1989) A small scale procedure for the rapid isolation of plant RNAs. Nuc. Acids Res. 17: 2362 

Transcription profiling time series of leaves from winter wheat grown under S and N-deficient conditions

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