5 protocols
Agilent G2565AA and G2565BA Microarray Scanner and Feature ExtractionVersion: 7.0,10.5Agilent publication number: G2566-90017, G4460-90019URL: http://www.chem.agilent.com/Library/usermanuals/Public/G2566-90017.pdf, http://www.chem.agilent.com/Library/usermanuals/Public/G4460-90019_FE_10.5_User.pdfScanner manual for software release 7.0. The Feature Extraction Software User Guide shows you how to set up and run Feature Extraction automatically for a batch of image files and how to extract image files in real time.
nucleic acid hybridization to array protocol
Overall 25,262 transcripts were represented on the microarray by 1, 2 or 3 individual probes. Hybridizations for all experimental conditions were performed in 4 replicates. Total RNA samples derived from the treatments were hybridized against the pooled control respective to their origin. The microarray hybridization procedure was carried out with 300ng of cyanine-3 and cyanine-5 labelled cRNA for 17h at 65°C. Control/control hybridization were performed, each component of the pooled control (LP 2°C, LP 7°C, LP 12°C) was hybridized against the pooled control to mitigate dye bias effects. Subsequently microarray disassembly and wash procedure followed as described by the manufacturer’s instructions (Agilent).
Agilent Two-Color Microarray-Based Gene Expression (Quick Amp Labeling)
Version: 5.7
Agilent publication number: G4140-90050
URL: http://www.chem.agilent.com/Library/usermanuals/Public/G4140-90050_Two-Color_GE_5.7.pdf
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.
The Ae. aegypti New Orleans (NO),Cayman and Cuba strains were reared under standard conditions (28 + 2 oC, 80 % RH) at the Liverpool School of Tropical Medicine. The NEW ORLEANS (NO) strain is a laboratory strain that is susceptible to all known insecticides which was originally colonized by the Center for Disease Control and Prevention (CDC) Atlanta, USA. The pyrethroid resistant CAYMAN strain was colonized from larvae collected in routine field surveillance sites in Grand Cayman in 2008. This strain has very high levels of resistance to DDT (>90% survival after 8 hours exposure to 4 % DDT) and pyrethroids (resistance ratio of 109-fold to permethrin and, 30-fold to deltamethrin using compared with the susceptible New Orleans as a susceptible strain). The CUBA-DELTA SAN 12 strain (CUBA-DELTA) was collected in 1997 in Santiago de Cuba. It was selected for 12 generations at the larval stage with deltamethrin at the Institute ‘Pedro Kouri’ in Havana, Cuba. CUBA-DELTA larvae were highly resistant to this insecticide (> 1000-fold) and this resistance was also manifested at the adult stage.
For each strain, total RNA was extracted from three pools of 30, three day old, non blood-fed females using Pico PureTM RNA Isolation Kit (Applied biosystems, Foster city, CA, USA). The strains were reared in parallel to minimize variation resulting from breeding conditions. Each biological replicate consisted of mosquitoes from distinct generations to control for stochastic variations. The quality and concentration of RNA was assessed using a 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA, USA).