Robotic Spotting

Applications

The full utility of the spotted microarray format is clearly reflected in the range of its applications. Transcriptome arrays, containing cDNA, gDNA, or oligonucleotide probes, are used to measure differential gene expression. Whole-genome arrays, typically composed of tiled gDNA or oligonucleotides, have been used to identify in vivo sites of protein-DNA interactions or allelic variation. Whilst these applications dominate, other formats, for example antibody arrays, facilitate analysis of protein and small-molecule analytes. Thus, spotted microarrays enable high-throughput, cost-effective, and large-scale analysis of molecular interactions.

Procedure

Robotic spotters deposit probes as an ordered array by repetition of a simple multi-step procedure. First, the print tool is positioned over the first batch of probes to be printed, which have previously been dissolved in spotting buffer and arranged in 96-, 384- or 1536-well microtitre plates. Second, the spotting pins are filled by capillary action with probe material for spotting. This step is often called a source visit. Third, the probe material is deposited on chemically-modified glass slides. Finally, the pins are cleaned before re-filling and printing the next batch of probes, to prevent cross-contamination between subsequent spot depositions. The diversity of instrumentation, spotting pins, and reagents available, mean that operators are able to refine their procedures for optimal throughput, spot density, morphology, and consistency. Whilst this facilitates production of high-quality arrays, it can also lead to significant differences between facilities with regard to the instrumentation, protocols, and reagents employed.

Limitations

Robotic spotters are supplied with sophisticated software to convert operator inputs to the precise list of instructions needed by the arrayer, e.g., how often each source visit is to be printed, and at which spot location. Most spotters, however, are not supplied with adequate array design tools. Operators are instead left to develop suitable spot layouts in an ad hoc fashion. This often leads to a sub-optimal designs with spots positioned according to print order, thus juxtaposing replicates, when a non-sequential or randomised spot layout would help to control for confounding spatial effects. For example, spatial biases caused by inconsistent probe concentrations in the microtitre plates and local variations in hybridisation or washing efficiency. Whilst random noise can be overcome with simple replication and averaging, systematic biases must be specifically addressed by randomisation and normalisation. Additionally, there is also a need for a tool that enables use of variable numbers of replicates, as current spot density constraints and the need for genome-wide coverage, mean that replication is often limited to the normalisation controls. Exogenous or 'spike' controls, i.e., probes that are complementary to targets not present in the genome of interest, can be employed for this purpose. Print time and maxmimum meta-grid area estimates enable users to evaluate the suitability of the array design.

SimArray

Simarray addresses the need for a user-friendly and user-configurable program that generates randomised spot maps, computes the maximum meta-grid area, and estimates the print time, in response to user-defined design decisions. The user enters these parameters by running SimArray twice. The first run produces the source visit list that can be edited to include variable numbers of replicates, or for specific source vists to be ommitted when plates are partially filled. The second tun processes this source list to create the spot layout, maximum medta-gid area and estimated print time. User configurable operation means that SimArray can be adaopted to most production environments.