- Microarray technology provides an unprecedented and uniquely comprehensive probe into the coordinated workings of entire biological pathways and genomic-level processes.
- In general terms, microarrays refer to a variety of platforms in which high density assays are performed in parallel on a solid support.
- Three major types of microarrays exist — tissue, protein, and DNA.
- Tissue microarrays immobilize small amounts of tissue from biopsies of multiple subjects on glass slides for immunohistochemical processing, while protein arrays immobilize peptides or intact proteins for detection by antibodies or other means.
AUTOANTIGEN AND CYTOKINE MICROARRAYS
- Applications of protein microarrays include assessment of enzyme–substrate,
protein–protein, and DNA–protein interactions.
- Proteins consist of highly diverse conformational structures that result from 20 amino acids vs. the 4 nucleic acid building blocks that generate a relatively uniform structure in DNA. Proteins may exist as large complexes, can be hydrophilic or hydrophobic, acidic or basic, and contain post-translational modifications such as acetylation, glycosylation, or phosphorylation.
- Functional and conformational properties of proteins must often remain intact
when immobilized onto a microarray in order to retain the desired binding properties for detection of target ligands.
- The development of protein microarrays to detect immunologic targets such
as cytokines or autoantibodies has enormous potential for research and diagnostic applications in autoimmune diseases.
- Microarrays that simultaneously detect multiple cytokines have been developed.
- Their method utilizes capture antibodies spotted onto membranes, incubation with biological samples such as patient serum, and detection by biotin-conjugated antibodies and enzymatic-coupled enhanced chemiluminescence.
- Twenty-eight cytokines were detected using this DNA Microarrays and Related Genomics Techniques method, including interleukins-1α, 2, 3, 5, 6, 7, 8, 10, 13, and 15; tumor necrosis factors α, and β; interferon-γ, and others.
- In addition to detecting multiple cytokines simultaneously, these assays were shown to be more sensitive than conventional ELISAs, with broader detection ranges.
- The ability to readily scale up this approach to include much larger numbers of cytokines and other proteins will undoubtedly fuel further development of this powerful tool for studying complex and dynamic cellular processes such as immune reactions, apoptosis, cell proliferation, and differentiation.
DNA AND OLIGONUCLEOTIDE MICROARRAYS
- DNA microarrays were first introduced in the mid-1990s and have been the
most widely utilized application of microarray technology.
- There are two commonly available DNA microarray systems. First are the cDNA microarrays fabricated by robotic spotting of PCR products, derived primarily from the 3′ end of genes and expressed sequence tags (ESTs), onto glass slides — this is the method popularized.
- The second method uses in situ synthesized oligonucleotide arrays that are fabricated using photolithographic chemistry on silicon chips.
- A third method involves spotting previously synthesized longer (40 to 70mer) oligonucleotides on either glass ornylon and plastic.
- In the spotted cDNA and long oligo microarray systems, two probeswith different fluorescent tags are hybridized to the same array, one serving as the
experimental condition and the other as a control.
- The ratio of hybridization between the two probes is calculated, allowing a quantization of the hybridization signal for each spot on the array. In this system, the probe is 1st strand cDNA generated by oligo-dT primed reverse transcription from an RNA sample
- A single labeled probe is used and each gene on the chip is represented by 8 to
10 wild-type 25-mer oligonucletides and the same number of single base mutant 25-mer oligonucleotides synthesized next to one another on the array.
- Signal intensity and the ratio of specific to nonspecific hybridization allows the generation of quantitative data regarding gene expression in the sample