Grain Hardness

ABSTRACT: A microarray analysis of wheat grain hardness For both the Heron and Falcon NIL, whole wheat heads were harvested at 6, 15 and 25 days post anthesis (DPA) from glass house grown material and stored at -80 OC. For the other wheat cultivars analysed, heads at approximately 9-10 DPA were used. Eight to ten developing caryopses at the same age and morphological stage of development were selected from a head to represent a sample. Seed collected from a different head represented a replicate sample. Total RNA was isolated from the whole seed following the method of Higgins et al. (1976) with the following modification to remove starch. After the lithium chloride precipitation, the RNA pellet was resuspended in 250 ?l water. Then 3.5 ?l of 3 M sodium acetate (pH 5.3) and 125 ?l ethanol were added and the sample was centrifuged for 10 minutes at 4 OC. The supernatant was transferred to a fresh tube and the RNA was ethanol-precipitated by the addition of 21.5 ?l of 3 M sodium acetate (pH 5.3) and 375 ?l ethanol. The recovery of RNA varied from 0.06-0.5 ?g RNA per mg of tissue and this was dependent on the developmental stage of the seeds used for extraction of the RNA. For the labelling procedure, 50 ?g of total RNA was used for both the Cy3 and Cy5 dyes (Amersham Pharmacia, UK), following the two-step labelling method of Schenk et al. (2000). The Cy3 control (hard wheat) and Cy5 sample (soft wheat) were swapped among the replicate experiments to minimise any bias in cDNA incorporation and photo-bleaching of the fluorescent dyes. The pre-hybridisation of the microarray slides, hybridisation of the target cDNA and subsequent washing of the slides to remove unbound target was performed as per the supplied protocol for the CMT-GAPSTM coated microscope slides (Corning USA). The slides were scanned with a GenePix 4000A microarray scanner (Axon Instruments, Union, CA, USA). The features were analysed using the GenePix Pro 4 software and unsatisfactorily segmented features were either manually adjusted or discarded to ensure the integrity of the data obtained.

ORGANISM(S): Triticum aestivum

SUBMITTER: Gavin Kennedy 

PROVIDER: E-GEOD-5369 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress