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''Two-color microarrays'' or ''two-channel microarrays'' are typically [[DNA hybridization|hybridized]] with cDNA prepared from two samples to be compared (e.g. diseased tissue versus healthy tissue) and that are labeled with two different [[fluorophore]]s.<ref name="Shalon et al.">{{cite journal|author=Shalon D|author2=Smith SJ|author3=Brown PO|date= 1996|title=A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization|journal=Genome Res|volume=6|pages=639–645|pmid=8796352|doi=10.1101/gr.6.7.639|issue=7|doi-access=free}}</ref> [[Fluorescence|Fluorescent]] dyes commonly used for cDNA labeling include [[Cyanine|Cy]]3, which has a fluorescence emission wavelength of 570 nm (corresponding to the green part of the light spectrum), and [[Cyanine|Cy]]5 with a fluorescence emission wavelength of 670 nm (corresponding to the red part of the light spectrum). The two Cy-labeled 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 [[Excited state|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.<ref name="Tang et al.">{{cite journal|author=Tang T|author2=François N|author3=Glatigny A|author4=Agier N|author5=Mucchielli MH|author6=Aggerbeck L|author7=Delacroix H|date= 2007|title=Expression ratio evaluation in two-colour microarray experiments is significantly improved by correcting image misalignment|journal=Bioinformatics|volume=23|pages=2686–2691|pmid=17698492|doi=10.1093/bioinformatics/btm399|issue=20|doi-access=free}}</ref>
Oligonucleotide microarrays often carry control probes designed to hybridize with [[RNA spike-in]]s. The degree of hybridization between the spike-ins and the control probes is used to [[Normalization (statistics)|normalize]] the hybridization measurements for the target probes. Although absolute levels of gene expression may be determined in the two-color array in rare instances, the relative differences in expression among different spots within a sample and between samples is the preferred method of [[data analysis]] for the two-color system. Examples of providers for such microarrays includes [[Agilent]] with their Dual-Mode platform, [[Eppendorf (company)|Eppendorf]] with their
In ''single-channel microarrays'' or ''one-color microarrays'', the arrays provide intensity data for each probe or probe set indicating a relative level of hybridization with the labeled target. However, they do not truly indicate abundance levels of a gene but rather relative abundance when compared to other samples or conditions when processed in the same experiment. Each RNA molecule encounters protocol and batch-specific bias during amplification, labeling, and hybridization phases of the experiment making comparisons between genes for the same microarray uninformative. The comparison of two conditions for the same gene requires two separate single-dye hybridizations. Several popular single-channel systems are the Affymetrix "Gene Chip", Illumina "Bead Chip", Agilent single-channel arrays, the Applied Microarrays "
One channel microarray may be the only choice in some situations. Suppose <math>i</math> samples need to be compared: then the number of experiments required using the two channel arrays quickly becomes unfeasible, unless a sample is used as a reference.
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