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])
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.
nucleic acid extraction protocol
For each strain, total RNA was extracted from three pools of 8-10 one to five day old, non blood-fed female Anopheles using the Ambion RNA-4-PCR Kit. RNA was treated with DNaseI to remove any contaminating DNA. The quality and concentration of RNA was assessed using a 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA, USA).