Some publications indicate that in-house spotted microarrays may not provide the same level of sensitivity as to commercial oligonucleotide arrays, possibly owing to the small batch sizes and reduced printing efficiencies. There is a company who offers a commercial array platform where 30-mer oligonucleotide probes (sequences of 30 nucleotides in length) are piezoelectrically deposited on an acrylamide matrix without any contact being made between the depositing equipment and the array surface itself. These arrays are comparable in quality to most manufacturers' arrays and generally superior to in-house printed arrays.
In oligonucleotide microarrays,
the probes are short sequences designed to match parts of the sequence of known or predicted open reading frames. Although oligonucleotide probes are often used in "spotted" microarrays, the term "oligonucleotide array" most often refers to a specific technique. Oligonucleotide arrays are produced by printing short oligonucleotide sequences designed to represent a single gene or family of gene splice-variants by synthesizing this sequence directly onto the array surface instead of depositing intact sequences. Sequences may be longer (60-mer probes) or shorter (25-mer probes) depending on the desired purpose; longer probes are more specific to individual target genes, and shorter probes may be spotted across the array in higher densities and are cheaper to manufacture.
One technique used to produce oligonucleotide arrays involves photolithographic synthesis on a silica substrate where light and light-sensitive masking agents are used to build a sequence one nucleotide at a time across the entire array. Each applicable probe is selectively unmasked prior to bathing the array in a solution containing just one nucleotide. Then a masking reaction takes place and the next set of probes are unmasked in preparation for a different nucleotide exposure. After many repetitions, the sequence of every probe becomes complete. More recently, Maskless Array Synthesis from NimbleGen Systems has adapted the flexibility of this system with other methods to produce larger numbers of probes.
Two-color vs. one-color detection
Two-color microarrays are typically hybridized with cDNA prepared from two samples that the researchers wish to compare (such as diseased tissue versus healthy tissue).These samples are labeled with two different fluorophores. Fluorescent dyes commonly used for cDNA labelling include Cy3, which has a fluorescence emission wavelength of 570 nm (corresponding to the green part of the light spectrum), and Cy5 with a fluorescence emission wavelength of 670 nm (corresponding to the red part of the light spectrum). The two Cy-labelled cDNA samples are mixed and hybridized to a single microarray that is then scanned in a microarray scanner to visualize fluorescence of the two fluorophores after excitation with a laser beam of a defined wavelength. Relative intensities of each fluorophore may then be used in ratio-based analysis to identify up-regulated and down-regulated genes.
Oligonucleotide microarrays often contain control probes designed to hybridize with RNA spike-ins. The degree of hybridization between the spike-ins and the control probes is used to normalize the hybridization measurements for the target probes. Although absolute levels of gene expression may be determined using the two-color array system, the system is more apt for the determination of relative differences in expression among different spots within a sample and between samples.
Single-channel microarrays, also called one-color microarrays, are designed to give estimations of the absolute levels of gene expression. Therefore, the comparison of the two sets of conditions requires two separate single-dye hybridizations. Since only a single dye is used, the data collected represent absolute values of gene expression. These may be compared to other genes within a sample or to reference normalizing probes used to calibrate data across the entire array and across multiple arrays. One strength of the single-dye system lies in the fact that an aberrant sample cannot affect the raw data derived from other samples, because each array chip is exposed to only one sample (as opposed to a two-color system, in which a single low-quality sample may drastically impinge on overall data precision even if the other sample is of high quality). Another benefit is that data are more easily compared to arrays from different experiments; the absolute values of gene expression may be compared between studies conducted months or years apart. One drawback to the one-color system, however, is that, when compared to the two-color system, twice as many microarrays are needed to compare samples within an experiment.