6 protocols
array scanning and feature extraction protocol
Feature extraction performed as recommended by manufacturer.
nucleic acid hybridization to array protocol
Hybridisation and washing procedures as described In manufacturer's protocol, pp 19-24.
Scanning performed as recommended by the manufacturer (Agilent G2505B).
Wild type A. thaliana accession Columbia 0 (Col0) were grown in controlled environment cabinets under a light intensity of 250 ?mol m -2 s -1, with a 10h photoperiod at a constant temperature of 22 C and a relative humidity of 70 %. Green peach aphid (Myzus persicae Sulzer) had been collected in Scotland in the years 2002-2004 and assigned genotype G (Kasprowicz et al., 2008). Aphids were maintained in an insectiary on mature potato plants in transparent Perspex cages with a 16h photoperiod and day/night temperature of 18 C. One mature fully expanded rosette leaf per plant was enclosed in a 2.5 cm mesh covered clip cage. At the start of the experiment all plants to be used were caged and 60 adult wingless aphids were confined to 4 cages. Following 24 h, 60 adult aphids were placed within each of a further 4 clip cages and after a further 18 h, 60 adult aphids were added to each of a further 4 clip cages. 6h after the final addition of aphids, all clip cages were removed and aphids were quickly brushed from leaves using a fine paint brush. Both the caged leaf (local leaf) and a second mature fully expanded rosette leaf immediately opposite the caged leaf (systemic leaf) were removed with a scalpel, transferred to 1.5 ml eppendorf tubes and rapidly frozen in liquid N2. Controls for the caged and non-caged leaves were collected from plants that had empty cages on for the 48 hours of the experiment. All leaves were harvested at the same time.
RNA was isolated from individual Arabidopsis leaves using the Qiagen RNeasy Plant Mini kit (Qiagen) according to the manufacturer's protocol and the quality of RNA was assessed using Agilent 2100 Bioanalyzer.
Target RNA (up to 100 ng) was labeled as recommended (version 6.5).