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Chapter 54 : Microarrays: Design and Use for Agricultural and Environmental Applications
There are two primary types of microarrays, cDNA-based and oligonucleotide-based microarrays. This chapter focuses primarily on oligonucleotide-based technology because of the specialized problems encountered in agricultural and environmental microbiology. In general, oligonucleotide microarrays are designed by using some of the basic principles of primer and probe design for PCR and Southern hybridization. Microarrays targeting mRNA enable researchers to relate community structure to community function. The design of these types of microarrays allows investigators to evaluate gene expression and therefore to expose important metabolic activities of specific microbial communities. There are several issues related to the use of microarrays that limit their use in environmental studies. At the front end of this potentially powerful tool is the need for sensitive nucleic acid extraction from complicated sample matrices. Direct extraction of nucleic acids from environmental samples may coextract humic acids or other organic materials that may affect nucleic acid hybridization with microarrays. PCR amplification has been used by several groups to amplify nucleic acids prior to microarray analysis. In situations where uncharacterized organisms are present, exhaustive gene or clone libraries for the target gene need to be gathered in order to design group- or clade-specific probes for detection and distinction among all possible targets within the studied ecosystem.
Schematic representation of a microarray experiment using the two-dye cross-hybridization technique. In this experiment, the expression profiles of a control cell and a cell of the same genetic lineage that has been exposed to a treatment are compared. The RNAs from the control are labeled during cDNA formation with Cy5, which is artificially colored red (note that the figure is printed in black and white and meant only to provide a schematic concept of the experimental procedure). The RNAs from the treated cells are extracted and labeled with Cy3 (green) during cDNA synthesis. After being labeled, the two samples are cohybridized to the microarray. Those genes that are expressed in more abundance in the control sample (or down-regulated during treatment) will show up as green spots. Conversely, those genes that are expressed in more abundance in the treatment sample will be visualized as red spots. Those genes which are expressed equally in the treatment and control samples will appear as yellow spots. In this way, one can see how the expression of an entire transcriptome responds to experimental treatments.
Two different slides printed and evaluated by use of a Paragon DNA microarray quality control stain kit. The slide on the left has consistent well-defined spots, while the slide on the right appears to have either too little oligonucleotide applied or an improper chemistry mixture that does not allow the spots to set and fix uniformly. A proper spotting solution must be used with the proper slide chemistry to allow for uniform spotting quality.
Common microarray slide coatings and associated spotting solutions
Microarray equipment and reagent supply companies