is shown at bottom left for size comparison. Many types of arrays exist and the broadest distinction is whether they are spatially arranged on a surface or on coded beads: • The traditional solid-phase array is a collection of orderly microscopic "spots", called features, each with thousands of identical and specific probes attached to a solid surface, such as
glass,
plastic or
silicon biochip (commonly known as a
genome chip,
DNA chip or
gene array). Thousands of these features can be placed in known locations on a single DNA microarray. • The alternative bead array is a collection of microscopic polystyrene beads, each with a specific probe and a ratio of two or more dyes, which do not interfere with the fluorescent dyes used on the target sequence. DNA microarrays can be used to detect DNA (as in
comparative genomic hybridization), or detect RNA (most commonly as
cDNA after
reverse transcription) that may or may not be translated into proteins. The process of measuring gene expression via cDNA is called
expression analysis or
expression profiling. Applications include: Specialised arrays tailored to particular
crops are becoming increasingly popular in
molecular breeding applications. In the future they could be used to screen
seedlings at early stages to lower the number of unneeded seedlings tried out in breeding operations.
Fabrication Microarrays can be manufactured in different ways, depending on the number of probes under examination, costs, customization requirements, and the type of scientific question being asked. Arrays from commercial vendors may have as few as 10 probes or as many as 5 million or more micrometre-scale probes.
Spotted vs. in situ synthesised arrays at the
University of Delaware Microarrays can be fabricated using a variety of technologies, including printing with fine-pointed pins onto glass slides,
photolithography using pre-made masks, photolithography using dynamic micromirror devices, ink-jet printing, or
electrochemistry on microelectrode arrays. In
spotted microarrays, the probes are
oligonucleotides,
cDNA or small fragments of
PCR products that correspond to
mRNAs. The probes are
synthesized prior to deposition on the array surface and are then "spotted" onto glass. A common approach utilizes an array of fine pins or needles controlled by a robotic arm that is dipped into wells containing DNA probes and then depositing each probe at designated locations on the array surface. The resulting "grid" of probes represents the nucleic acid profiles of the prepared probes and is ready to receive complementary cDNA or cRNA "targets" derived from experimental or clinical samples. This technique is used by research scientists around the world to produce "in-house" printed microarrays in their own labs. These arrays may be easily customized for each experiment, because researchers can choose the probes and printing locations on the arrays, synthesize the probes in their own lab (or collaborating facility), and spot the arrays. They can then generate their own labeled samples for hybridization, hybridize the samples to the array, and finally scan the arrays with their own equipment. This provides a relatively low-cost microarray that may be customized for each study, and avoids the costs of purchasing often more expensive commercial arrays that may represent vast numbers of genes that are not of interest to the investigator. Publications exist which indicate in-house spotted microarrays may not provide the same level of sensitivity compared to commercial oligonucleotide arrays, possibly owing to the small batch sizes and reduced printing efficiencies when compared to industrial manufactures of oligo 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 of manufacturing. 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 such as the
Agilent design) or shorter (25-mer probes produced by
Affymetrix) depending on the desired purpose; longer probes are more specific to individual target genes, shorter probes may be spotted in higher density across the array and are cheaper to manufacture. One technique used to produce oligonucleotide arrays include
photolithographic synthesis (Affymetrix) 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 of a single 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 sequences of every probe become fully constructed. More recently, Maskless Array Synthesis from NimbleGen Systems has combined flexibility with large numbers of probes.
Two-channel vs. one-channel detection Two-color microarrays or
two-channel microarrays are typically
hybridized with cDNA prepared from two samples to be compared (e.g. diseased tissue versus healthy tissue) and that are labeled with two different
fluorophores.
Fluorescent dyes commonly used for cDNA labeling 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-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
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 carry 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 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 with their DualChip platform for colorimetric
Silverquant labeling, and TeleChem International with
Arrayit. 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 "CodeLink" arrays, and the Eppendorf "DualChip & Silverquant". 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 was of high quality). Another benefit is that data are more easily compared to arrays from different experiments as long as batch effects have been accounted for. One channel microarray may be the only choice in some situations. Suppose i 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.
A typical protocol across the array indicates the abundance of a predetermined set of sequences. These sequences are typically specifically chosen to report on genes of interest within the organism's genome. This is an example of a
DNA microarray experiment which includes details for a particular case to better explain DNA microarray experiments, while listing modifications for RNA or other alternative experiments. • The two samples to be compared (pairwise comparison) are grown/acquired. In this example treated sample (
case) and untreated sample (
control). • The
nucleic acid of interest is purified: this can be
RNA for
expression profiling,
DNA for
comparative hybridization, or DNA/RNA bound to a particular
protein which is
immunoprecipitated (
ChIP-on-chip) for
epigenetic or regulation studies. In this example total RNA is isolated (both nuclear and
cytoplasmic) by
guanidinium thiocyanate-phenol-chloroform extraction (e.g.
Trizol) which isolates most RNA (whereas column methods have a cut off of 200 nucleotides) and if done correctly has a better purity. • The purified RNA is analysed for quality (by
capillary electrophoresis) and quantity (for example, by using a
NanoDrop or NanoPhotometer
spectrometer). If the material is of acceptable quality and sufficient quantity is present (e.g., >1
μg, although the required amount varies by microarray platform), the experiment can proceed. • The labeled product is generated via
reverse transcription and followed by an optional
PCR amplification. The RNA is reverse transcribed with either polyT primers (which amplify only
mRNA) or random primers (which amplify all RNA, most of which is
rRNA).
miRNA microarrays ligate an oligonucleotide to the purified small RNA (isolated with a fractionator), which is then reverse transcribed and amplified. • The label is added either during the reverse transcription step, or following amplification if it is performed. The
sense labeling is dependent on the microarray; e.g. if the label is added with the RT mix, the
cDNA is antisense and the microarray probe is sense, except in the case of negative controls. • The label is typically
fluorescent; only one machine uses
radiolabels. • The labeling can be direct (not used) or indirect (requires a coupling stage). For two-channel arrays, the coupling stage occurs before hybridization, using
aminoallyl uridine triphosphate (aminoallyl-UTP, or aaUTP) and
NHS amino-reactive dyes (such as
cyanine dyes); for single-channel arrays, the coupling stage occurs after hybridization, using
biotin and labeled
streptavidin. The modified nucleotides (usually in a ratio of 1 aaUTP: 4 TTP (
thymidine triphosphate)) are added enzymatically in a low ratio to normal nucleotides, typically resulting in 1 every 60 bases. The aaDNA is then purified with a
column (using a phosphate buffer solution, as
Tris contains amine groups). The aminoallyl group is an amine group on a long linker attached to the nucleobase, which reacts with a reactive dye. • A form of replicate known as a dye flip can be performed to control for dye
artifacts in two-channel experiments; for a dye flip, a second slide is used, with the labels swapped (the sample that was labeled with Cy3 in the first slide is labeled with Cy5, and vice versa). In this example,
aminoallyl-UTP is present in the reverse-transcribed mixture. • The labeled samples are then mixed with a proprietary
hybridization solution which can consist of
SDS,
SSC,
dextran sulfate, a blocking agent (such as
Cot-1 DNA, salmon sperm DNA, calf thymus DNA,
PolyA, or PolyT),
Denhardt's solution, or
formamine. • The mixture is denatured and added to the pinholes of the microarray. The holes are sealed and the microarray hybridized, either in a hyb oven, where the microarray is mixed by rotation, or in a mixer, where the microarray is mixed by alternating pressure at the pinholes. • After an overnight hybridization, all nonspecific binding is washed off (SDS and SSC). • The microarray is dried and scanned by a machine that uses a laser to excite the dye and measures the emission levels with a detector. • The image is gridded with a template and the intensities of each feature (composed of several pixels) is quantified. • The raw data is normalized; the simplest normalization method is to subtract background intensity and scale so that the total intensities of the features of the two channels are equal, or to use the intensity of a reference gene to calculate the
t-value for all of the intensities. More sophisticated methods include
z-ratio,
loess and lowess regression and RMA (robust multichip analysis) for Affymetrix chips (single-channel, silicon chip,
in situ synthesized short oligonucleotides). == Microarrays and bioinformatics ==