ABSTRACT: Genomic approaches to the discovery of promoters for sustained expression in cotton (Gossypium hirsutum L.) under field conditions: expression analysis in transgenic cotton and Arabidopsis of a Rubisco small subunit promoter identified using EST sequence analysis and cDNA microarrays. RNAs from 8-week old plants were compared to RNAs from 10-, 14-, 20- and 22-week old plants on separate arrays each with a dye swap. The 20 and 22 weeks hybridisations each had a biological replicate (each with a dye swap) as well as a second independent hybridisation (with dye swap) with one of the 22 week RNAs. One of the 22-week slides was of low quality and had to be discarded.
Project description:Cycloheximide treatment of cotton ovules Keywords: Antibiotic Treatment Single timepoint, 6 slides, 3 Biological replicates, each biological replicate having 2 technical replicates. Dyes were swapped between technical replicates.
Project description:Cotton ovule development, mutant vs wild type, Comparisons of DP16 0 dpa ovule Keywords: WildType vs Mutant 7 comparisons and one 0 dpa control. The 1A/DP16 & 4A/DP16 comparisons have 8 slides each, 4 Biological replicates, each biological replicate having 2 technical replicates. Dyes were swapped between technical replicates. The SL1-7-1/DP16 comparison has 6 slides, 3 Biological replicates, each biological replicate having 2 technical replicates. Dyes were swapped between technical replicates. The 5B/DP16, fl/Xu-142, OI/II+N & 53/DP16 comparisons have 4 slides each, 2 Biological replicates, each biological replicate having 2 technical replicates. Dyes were swapped between technical replicates. The 0 dpa control experiment has 3 slides , 3 Biological replicates, no technical replicates and no dye swapping.
Project description:DP16 ovules of -4, -2, 2 dpa compared to 0 dpa Keywords: Time Course 4 timepoints at -4 dpa, -2 dpa, 0 dpa and +2 dpa. -4, -2 and +2 dpa have 4 slides each, 2 Biological replicates, each biological replicate having 2 technical replicates. Dyes were swapped between technical replicates. 0 dpa has 3 slides , 3 Biological replicates,no technical replicates and no dye swapping.
Project description:Himalaya v 292 Four biological replicates were used for the comparison and the dyes were swapped among the test and control samples for labeling two of the replicates. For each replicate, 50 ?g of total RNA was used for both the Cy3 and Cy5 dyes (Amersham Pharmacia, UK), following the two-step labeling method of Schenk et al. (2000). The control plant, 'Himalaya' was labeled with Cy3 in the un-swapped replicates. The dyes were reversed among the replicate experiments to minimize any bias in cDNA incorporation and photo-bleaching of the fluorescent dyes. The pre-hybridization of the microarray slides, hybridization of samples 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 analyzed using the GenePix Pro 4 software and unsatisfactorily segmented features were either manually adjusted or discarded to ensure the integrity of the data obtained.
Project description: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.
Project description:0dpa Fibre vs epidermal laser capture microdisection comparison Total RNA was isolated from each cell type (fibre initial or non-fibre epidermal pavement cell) from both Plant 1 and Plant 2. Hence, there were two biological replicates of each cell-type. Sub-samples of each cell type, designated here as technical replicates, were taken as separate batches of cells from different groups of ovules, but each from the same pair of plants. In particular, there were four sub-samples of fibre initial cells from plant 1 and two from plant 2 and two sub-samples of non-fibre epidermal from plant 1 and four from plant 2. The complete experiment involved three dye-swap pairs using a total of 6 arrays.
Project description:Comparison of pre and post metamorphis phases of cane toad Keywords: Developmental Stages Samples were harvested at 9, 18, 28 (pre-metamorph), 30 and 53 (post-metamorph) days of age and RNA extracted using Trizol Reagent (Invitrogen). Total RNA (100 mg) was reverse transcribed (Qiagen) and labelled cDNA probes were generated using the fluorophores, Cy3 and Cy5 (Amersham Pharmacia Biotech). Prehybridization of slides, application of the probe to the microarray slides, hybridisation, and subsequent washing steps were performed according to manufacturer's instructions (Corning Microarray Technology). Slides were scanned and analysed with a GenePix 4000B laser scanner and GenePix 3.0 and 4.0 software (Axon Instruments).