Agilent Two-Color Microarray-Based Gene Expression (Quick Amp Labeling)
Agilent publication number: G4140-90050
This document describes Agilent's recommended procedures for the preparation and labeling of complex biological targets and hybridization, washing, scanning, and feature extraction of Agilent 60-mer oligonucleotide microarrays for microarray-based two-color gene expression analysis.
Manufacturer's recommendations. (Parameters: Scanning hardware = G2505A DNA microarray scanner [Agilent], Scanning software = Feature Extraction Software [Agilent])
Arcturus picopure kit. 10 mosquitoes per extraction ground together. Manufacturer's protocol followed. On-column DNase digestion using Qiagen RNase-free DNase. Total RNA quantity measured by Nanodrop and quality with Agilent RNA 6000 Nano assay on Agilent 2100 Bioanalyser. Only total RNA with minimal degradation used for labelling. Parameters: Extracted product = total_RNA, Amplification = none)
Mosquitoes raised from field collected larvae or from colonies. Larvae raised at 2-300 per tray and fed tetramin fish food. Upon eclosion adults provided 10% sugar water (Parameters: time unit = seconds, temperature unit = C)
Manufacturer's recommendations. (Parameters: Chamber type = OTHER: Agilent, Quantity of label target used = 300, Mass unit = Nano gram, time = 17, Tiny time unit = hours, Volume = 50, Volume unit = Micro litre, temperature = 65)
One to five day old mosquitoes were exposed to either WHO insecticide papers or non-treated control papers in standard WHO susceptibility test kits. Exposure was for one hour before adults were transferred to holding tubes for a further 24 hours.
Anopheles larvae were collected from breeding sites and reared in plastic bowls in the insectaries at the Zanzibar Malaria Control Programme. Adults emerged from pupae in cages and were allowed to feed ad libitum on 20% sugar. Larvae were maintained on tetramin fish food.