Pivotal to the technical capacity of the ACGT Microarray Facility is the Generation III Spotter from Molecular Dynamics (GE Healthcare). The spotter is a precision robotic instrument capable of printing nanolitre volumes of nucleic acid samples onto glass slides, with an accuracy of 1 micron. Each sample typically represents an individual gene, and a maximum of 9000 spots can be placed on a single 2.5 x 7.5cm slide. As each target is printed in duplicate, this means that slides can be produced at the facility that allow the expression levels of over 4500 genes to be analysed simultaneously. Alternatively a unique spotting layout can be used, resulting in ~10000 unique targets printed on each slide.
The print head of the Generation III spotter consists of 12 stainless steel capillary pens that each load about 200nl of sample at a time - enough to print 36 slides without having to reload from the sample plate.
Hybridisation chambers are sealed compartments into which microarray slides are placed while the nucleic acid probe in solution hybridises to target spots on the slide. The purpose of the chamber is to create a microenvironment offering the correct humidity and temperature for the hybridisation reaction to occur. The ACGT Microarray Facility makes use of a locally manufactured hybridisation chamber, called HYB-UP for performing hybridisations in a waterbath.
Agilent hybridization oven
The Agilent hybridization oven of the Facility is used in conjunction with the in situ synthesized Agilent slides to ensure the highest quality data. The rotating oven allows for the uniform binding of probe mixture to the targets on the slide, enabling a higher relative intensity for low abundance transcripts which might not be resolved using conventional spotted arrays. The optimized Agilent reagents eliminate the need for hybridization optimizations, and dramatically decreases the non-specific background binding, resulting in a significantly higher signal-to-noise ratio.
Successful image acquisition of hybridized microarray slides is necessary before any analysis can be performed. For this purpose the ACGT Microarray Facility uses GenePix software, and the Axon 4000B scanner - one of the fastest and smallest scanners available. It is able to scan at a resolution of 5 microns, and has dual filters able to simultaneously detect emission wavelengths of 532nm (Cy3) and 635nm (Cy5), greatly reducing scanning time.