cDNA microarray
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A DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the
expression Expression may refer to: Linguistics * Expression (linguistics), a word, phrase, or sentence * Fixed expression, a form of words with a specific meaning * Idiom, a type of fixed expression * Metaphorical expression, a particular word, phrase, o ...
levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Each DNA spot contains picomoles (10−12
moles Moles can refer to: * Moles de Xert, a mountain range in the Baix Maestrat comarca, Valencian Community, Spain * The Moles (Australian band) *The Moles, alter ego of Scottish band Simon Dupree and the Big Sound People *Abraham Moles, French engin ...
) of a specific DNA sequence, known as '' probes'' (or ''reporters'' or '' oligos''). These can be a short section of a
gene In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...
or other DNA element that are used to hybridize a cDNA or cRNA (also called anti-sense RNA) sample (called ''target'') under high-stringency conditions. Probe-target hybridization is usually detected and quantified by detection of
fluorophore A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...
-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target. The original nucleic acid arrays were macro arrays approximately 9 cm × 12 cm and the first computerized image based analysis was published in 1981. It was invented by Patrick O. Brown. An example of its application is in SNPs arrays for polymorphisms in cardiovascular diseases, cancer, pathogens and GWAS analysis. It is also used for the identification of structural variations and the measurement of gene expression.


Principle

The core principle behind microarrays is hybridization between two DNA strands, the property of complementary nucleic acid sequences to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs. A high number of complementary base pairs in a nucleotide sequence means tighter
non-covalent In chemistry, a non-covalent interaction differs from a covalent bond in that it does not involve the sharing of electrons, but rather involves more dispersed variations of electromagnetic interactions between molecules or within a molecule. The ...
bonding between the two strands. After washing off non-specific bonding sequences, only strongly paired strands will remain hybridized. Fluorescently labeled target sequences that bind to a probe sequence generate a signal that depends on the hybridization conditions (such as temperature), and washing after hybridization. Total strength of the signal, from a spot (feature), depends upon the amount of target sample binding to the probes present on that spot. Microarrays use relative quantitation in which the intensity of a feature is compared to the intensity of the same feature under a different condition, and the identity of the feature is known by its position.


Uses and types

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 Glass is a non-crystalline, often transparent, amorphous solid that has widespread practical, technological, and decorative use in, for example, window panes, tableware, and optics. Glass is most often formed by rapid cooling ( quenching ...
,
plastic Plastics are a wide range of synthetic or semi-synthetic materials that use polymers as a main ingredient. Their plasticity makes it possible for plastics to be moulded, extruded or pressed into solid objects of various shapes. This adaptab ...
or
silicon Silicon is a chemical element with the symbol Si and atomic number 14. It is a hard, brittle crystalline solid with a blue-grey metallic luster, and is a tetravalent metalloid and semiconductor. It is a member of group 14 in the periodic ta ...
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 Comparative genomic hybridization (CGH) is a molecular cytogenetic method for analysing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells. The ...
), 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 In the field of molecular biology, gene expression profiling is the measurement of the activity (the expression) of thousands of genes at once, to create a global picture of cellular function. These profiles can, for example, distinguish between c ...
. Applications include: Specialised arrays tailored to particular
crop A crop is a plant that can be grown and harvested extensively for profit or subsistence. When the plants of the same kind are cultivated at one place on a large scale, it is called a crop. Most crops are cultivated in agriculture or hydropon ...
s are becoming increasingly popular in
molecular breeding Molecular breeding is the application of molecular biology tools, often in plant breedingVoosen P (2009Molecular Breeding Makes Crops Hardier and More Nutritious Markers, knockouts and other technical advances improve breeding without modifying gen ...
applications. In the future they could be used to screen
seedling A seedling is a young sporophyte developing out of a plant embryo from a seed. Seedling development starts with germination of the seed. A typical young seedling consists of three main parts: the radicle (embryonic root), the hypocotyl (embryo ...
s 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

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 Electrochemistry is the branch of physical chemistry concerned with the relationship between electrical potential difference, as a measurable and quantitative phenomenon, and identifiable chemical change, with the potential difference as an outco ...
on microelectrode arrays. In ''spotted microarrays'', the probes are
oligonucleotide Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids ...
s, cDNA or small fragments of PCR products that correspond to
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during the ...
s. 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 frame In molecular biology, open reading frames (ORFs) are defined as spans of DNA sequence between the start and stop codons. Usually, this is considered within a studied region of a prokaryotic DNA sequence, where only one of the six possible readin ...
s. 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 Agilent Technologies, Inc. is an American life sciences company that provides instruments, software, services, and consumables for the entire laboratory workflow. Its global headquarters is located in Santa Clara, California. Agilent was establi ...
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 In integrated circuit manufacturing, photolithography or optical lithography is a general term used for techniques that use light to produce minutely patterned thin films of suitable materials over a substrate, such as a silicon wafer, to protect ...
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
fluorophore A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...
s.
Fluorescent Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, ...
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 A laser is a device that emits light through a process of optical amplification based on the stimulated emission of electromagnetic radiation. The word "laser" is an acronym for "light amplification by stimulated emission of radiation". The fi ...
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 Agilent Technologies, Inc. is an American life sciences company that provides instruments, software, services, and consumables for the entire laboratory workflow. Its global headquarters is located in Santa Clara, California. Agilent was establi ...
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

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 In the field of molecular biology, gene expression profiling is the measurement of the activity (the expression) of thousands of genes at once, to create a global picture of cellular function. These profiles can, for example, distinguish between c ...
, DNA for comparative hybridization, or DNA/RNA bound to a particular
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
which is immunoprecipitated (
ChIP-on-chip ChIP-on-chip (also known as ChIP-chip) is a technology that combines chromatin immunoprecipitation ('ChIP') with DNA microarray (''"chip"''). Like regular ChIP, ChIP-on-chip is used to investigate interactions between proteins and DNA ''in vivo' ...
) for epigenetic or regulation studies. In this example total RNA is isolated (both nuclear and
cytoplasm In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. ...
ic) by
Guanidinium thiocyanate-phenol-chloroform extraction Acid guanidinium thiocyanate-phenol-chloroform extraction (abbreviated AGPC) is a liquid–liquid extraction technique in biochemistry. It is widely used in molecular biology for isolating RNA (as well as DNA and protein in some cases). This meth ...
(e.g.
Trizol TRIzol is a widely used chemical solution used in the extraction of DNA, RNA, and proteins from cells. The solution was initially used and published by Piotr Chomczyński and Nicoletta Sacchi in 1987. TRIzol is the brand name of guanidinium t ...
) 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 Capillary electrophoresis (CE) is a family of electrokinetic separation methods performed in submillimeter diameter capillaries and in micro- and nanofluidic channels. Very often, CE refers to capillary zone electrophoresis (CZE), but other elect ...
) and quantity (for example, by using a NanoDrop or NanoPhotometer
spectrometer A spectrometer () is a scientific instrument used to separate and measure spectral components of a physical phenomenon. Spectrometer is a broad term often used to describe instruments that measure a continuous variable of a phenomenon where the ...
). 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 In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during the ...
) or random primers (which amplify all RNA, most of which is
rRNA Ribosomal ribonucleic acid (rRNA) is a type of non-coding RNA which is the primary component of ribosomes, essential to all cells. rRNA is a ribozyme which carries out protein synthesis in ribosomes. Ribosomal RNA is transcribed from ribosoma ...
).
miRNA MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miR ...
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 A sense is a biological system used by an organism for sensation, the process of gathering information about the world through the detection of stimuli. (For example, in the human body, the brain which is part of the central nervous system re ...
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 Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, ...
; 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 Uridine (symbol U or Urd) is a glycosylated pyrimidine analog containing uracil attached to a ribose ring (or more specifically, a ribofuranose) via a β-N1-glycosidic bond. The analog is one of the five standard nucleosides which make up nucle ...
triphosphate (aminoallyl-UTP, or aaUTP) and
NHS The National Health Service (NHS) is the umbrella term for the publicly funded healthcare systems of the United Kingdom (UK). Since 1948, they have been funded out of general taxation. There are three systems which are referred to using the " ...
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 Tris, or tris(hydroxymethyl)aminomethane, or known during medical use as tromethamine or THAM, is an organic compound with the formula (HOCH2)3CNH2, one of the twenty Good's buffers. It is extensively used in biochemistry and molecular biology as ...
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 Polyadenylation is the addition of a poly(A) tail to an RNA transcript, typically a messenger RNA (mRNA). The poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In euka ...
, 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 In statistics, the ''t''-statistic is the ratio of the departure of the estimated value of a parameter from its hypothesized value to its Standard error (statistics), standard error. It is used in statistical hypothesis testing, hypothesis testin ...
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

The advent of inexpensive microarray experiments created several specific bioinformatics challenges: the multiple levels of replication in experimental design (
Experimental design The design of experiments (DOE, DOX, or experimental design) is the design of any task that aims to describe and explain the variation of information under conditions that are hypothesized to reflect the variation. The term is generally associ ...
); the number of platforms and independent groups and data format ( Standardization); the statistical treatment of the data ( Data analysis); mapping each probe to the
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during the ...
transcript that it measures (
Annotation An annotation is extra information associated with a particular point in a document or other piece of information. It can be a note that includes a comment or explanation. Annotations are sometimes presented in the margin of book pages. For anno ...
); the sheer volume of data and the ability to share it (
Data warehousing In computing, a data warehouse (DW or DWH), also known as an enterprise data warehouse (EDW), is a system used for reporting and data analysis and is considered a core component of business intelligence. DWs are central repositories of integr ...
).


Experimental design

Due to the biological complexity of gene expression, the considerations of experimental design that are discussed in the
expression profiling In the field of molecular biology, gene expression profiling is the measurement of the activity (the expression) of thousands of genes at once, to create a global picture of cellular function. These profiles can, for example, distinguish between c ...
article are of critical importance if statistically and biologically valid conclusions are to be drawn from the data. There are three main elements to consider when designing a microarray experiment. First, replication of the biological samples is essential for drawing conclusions from the experiment. Second, technical replicates (e.g. two RNA samples obtained from each experimental unit) may help to quantitate precision. The biological replicates include independent RNA extractions. Technical replicates may be two aliquots of the same extraction. Third, spots of each cDNA clone or oligonucleotide are present as replicates (at least duplicates) on the microarray slide, to provide a measure of technical precision in each hybridization. It is critical that information about the sample preparation and handling is discussed, in order to help identify the independent units in the experiment and to avoid inflated estimates of statistical significance.


Standardization

Microarray data is difficult to exchange due to the lack of standardization in platform fabrication, assay protocols, and analysis methods. This presents an
interoperability Interoperability is a characteristic of a product or system to work with other products or systems. While the term was initially defined for information technology or systems engineering services to allow for information exchange, a broader defi ...
problem in bioinformatics. Various
grass-roots A grassroots movement is one that uses the people in a given district, region or community as the basis for a political or economic movement. Grassroots movements and organizations use collective action from the local level to effect change at t ...
open-source projects are trying to ease the exchange and analysis of data produced with non-proprietary chips: For example, the "Minimum Information About a Microarray Experiment" (
MIAME Minimum information about a microarray experiment (MIAME) is a standard created by the FGED Society for reporting microarray experiments. MIAME is intended to specify all the information necessary to interpret the results of the experiment unam ...
) checklist helps define the level of detail that should exist and is being adopted by many journals as a requirement for the submission of papers incorporating microarray results. But MIAME does not describe the format for the information, so while many formats can support the MIAME requirements, no format permits verification of complete semantic compliance. The "MicroArray Quality Control (MAQC) Project" is being conducted by the US
Food and Drug Administration The United States Food and Drug Administration (FDA or US FDA) is a List of United States federal agencies, federal agency of the United States Department of Health and Human Services, Department of Health and Human Services. The FDA is respon ...
(FDA) to develop standards and quality control metrics which will eventually allow the use of MicroArray data in drug discovery, clinical practice and regulatory decision-making. The MGED Society has developed standards for the representation of gene expression experiment results and relevant annotations.


Data analysis

Microarray data sets are commonly very large, and analytical precision is influenced by a number of variables. Statistical challenges include taking into account effects of background noise and appropriate normalization of the data. Normalization methods may be suited to specific platforms and, in the case of commercial platforms, the analysis may be proprietary. Algorithms that affect statistical analysis include: * Image analysis: gridding, spot recognition of the scanned image (segmentation algorithm), removal or marking of poor-quality and low-intensity features (called ''flagging''). * Data processing: background subtraction (based on global or local background), determination of spot intensities and intensity ratios, visualisation of data (e.g. see
MA plot Within computational biology, an MA plot is an application of a Bland–Altman plot for visual representation of genomic data. The plot visualizes the differences between measurements taken in two samples, by transforming the data onto M (log ratio) ...
), and log-transformation of ratios, global or
local Local may refer to: Geography and transportation * Local (train), a train serving local traffic demand * Local, Missouri, a community in the United States * Local government, a form of public administration, usually the lowest tier of administrat ...
normalization of intensity ratios, and segmentation into different copy number regions using
step detection In statistics and signal processing, step detection (also known as step smoothing, step filtering, shift detection, jump detection or edge detection) is the process of finding abrupt changes (steps, jumps, shifts) in the mean level of a time seri ...
algorithms. * Class discovery analysis: This analytic approach, sometimes called unsupervised classification or knowledge discovery, tries to identify whether microarrays (objects, patients, mice, etc.) or genes cluster together in groups. Identifying naturally existing groups of objects (microarrays or genes) which cluster together can enable the discovery of new groups that otherwise were not previously known to exist. During knowledge discovery analysis, various unsupervised classification techniques can be employed with DNA microarray data to identify novel clusters (classes) of arrays. This type of approach is not hypothesis-driven, but rather is based on iterative pattern recognition or statistical learning methods to find an "optimal" number of clusters in the data. Examples of unsupervised analyses methods include self-organizing maps, neural gas, k-means cluster analyses, hierarchical cluster analysis, Genomic Signal Processing based clustering and model-based cluster analysis. For some of these methods the user also has to define a distance measure between pairs of objects. Although the Pearson correlation coefficient is usually employed, several other measures have been proposed and evaluated in the literature. The input data used in class discovery analyses are commonly based on lists of genes having high informativeness (low noise) based on low values of the coefficient of variation or high values of Shannon entropy, etc. The determination of the most likely or optimal number of clusters obtained from an unsupervised analysis is called cluster validity. Some commonly used metrics for cluster validity are the silhouette index, Davies-Bouldin index, Dunn's index, or Hubert's \Gamma statistic. * Class prediction analysis: This approach, called supervised classification, establishes the basis for developing a predictive model into which future unknown test objects can be input in order to predict the most likely class membership of the test objects. Supervised analysis for class prediction involves use of techniques such as linear regression, k-nearest neighbor, learning vector quantization, decision tree analysis, random forests, naive Bayes, logistic regression, kernel regression, artificial neural networks, support vector machines, mixture of experts, and supervised neural gas. In addition, various metaheuristic methods are employed, such as genetic algorithms, covariance matrix self-adaptation, particle swarm optimization, and
ant colony optimization In computer science and operations research, the ant colony optimization algorithm (ACO) is a probabilistic technique for solving computational problems which can be reduced to finding good paths through graphs. Artificial ants stand for multi ...
. Input data for class prediction are usually based on filtered lists of genes which are predictive of class, determined using classical hypothesis tests (next section), Gini diversity index, or information gain (entropy). * Hypothesis-driven statistical analysis: Identification of statistically significant changes in gene expression are commonly identified using the
t-test A ''t''-test is any statistical hypothesis test in which the test statistic follows a Student's ''t''-distribution under the null hypothesis. It is most commonly applied when the test statistic would follow a normal distribution if the value of ...
,
ANOVA Analysis of variance (ANOVA) is a collection of statistical models and their associated estimation procedures (such as the "variation" among and between groups) used to analyze the differences among means. ANOVA was developed by the statistician ...
,
Bayesian method Bayesian inference is a method of statistical inference in which Bayes' theorem is used to update the probability for a hypothesis as more evidence or information becomes available. Bayesian inference is an important technique in statistics, and e ...
Mann–Whitney test methods tailored to microarray data sets, which take into account
multiple comparisons In statistics, the multiple comparisons, multiplicity or multiple testing problem occurs when one considers a set of statistical inferences simultaneously or infers a subset of parameters selected based on the observed values. The more inferences ...
or cluster analysis. These methods assess statistical power based on the variation present in the data and the number of experimental replicates, and can help minimize
Type I and type II errors In statistical hypothesis testing, a type I error is the mistaken rejection of an actually true null hypothesis (also known as a "false positive" finding or conclusion; example: "an innocent person is convicted"), while a type II error is the fa ...
in the analyses. * Dimensional reduction: Analysts often reduce the number of dimensions (genes) prior to data analysis. This may involve linear approaches such as principal components analysis (PCA), or non-linear manifold learning (distance metric learning) using kernel PCA, diffusion maps, Laplacian eigenmaps, local linear embedding, locally preserving projections, and Sammon's mapping. * Network-based methods: Statistical methods that take the underlying structure of gene networks into account, representing either associative or causative interactions or dependencies among gene products. Weighted gene co-expression network analysis is widely used for identifying co-expression modules and intramodular hub genes. Modules may corresponds to cell types or pathways. Highly connected intramodular hubs best represent their respective modules. Microarray data may require further processing aimed at reducing the dimensionality of the data to aid comprehension and more focused analysis. Other methods permit analysis of data consisting of a low number of biological or technical replicates; for example, the Local Pooled Error (LPE) test pools standard deviations of genes with similar expression levels in an effort to compensate for insufficient replication.


Annotation

The relation between a probe and the
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during the ...
that it is expected to detect is not trivial. Some mRNAs may cross-hybridize probes in the array that are supposed to detect another mRNA. In addition, mRNAs may experience amplification bias that is sequence or molecule-specific. Thirdly, probes that are designed to detect the mRNA of a particular gene may be relying on genomic EST information that is incorrectly associated with that gene.


Data warehousing

Microarray data was found to be more useful when compared to other similar datasets. The sheer volume of data, specialized formats (such as
MIAME Minimum information about a microarray experiment (MIAME) is a standard created by the FGED Society for reporting microarray experiments. MIAME is intended to specify all the information necessary to interpret the results of the experiment unam ...
), and curation efforts associated with the datasets require specialized databases to store the data. A number of open-source data warehousing solutions, such as InterMine an
BioMart
have been created for the specific purpose of integrating diverse biological datasets, and also support analysis.


Alternative technologies

Advances in massively parallel sequencing has led to the development of RNA-Seq technology, that enables a whole transcriptome shotgun approach to characterize and quantify gene expression. Unlike microarrays, which need a reference genome and transcriptome to be available before the microarray itself can be designed, RNA-Seq can also be used for new model organisms whose genome has not been sequenced yet.


Glossary

* An ''array'' or ''slide'' is a collection of '' features'' spatially arranged in a two dimensional grid, arranged in columns and rows. * ''Block'' or ''subarray'': a group of spots, typically made in one print round; several subarrays/ blocks form an array. * ''Case/control'': an experimental design paradigm especially suited to the two-colour array system, in which a condition chosen as control (such as healthy tissue or state) is compared to an altered condition (such as a diseased tissue or state). * '' Channel'': the
fluorescence Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...
output recorded in the scanner for an individual
fluorophore A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...
and can even be ultraviolet. * ''Dye flip'' or ''dye swap'' or '' fluor reversal'': reciprocal labelling of DNA targets with the two dyes to account for dye bias in experiments. * ''Scanner'': an instrument used to detect and quantify the intensity of fluorescence of spots on a microarray slide, by selectively exciting fluorophores with a
laser A laser is a device that emits light through a process of optical amplification based on the stimulated emission of electromagnetic radiation. The word "laser" is an acronym for "light amplification by stimulated emission of radiation". The fi ...
and measuring the fluorescence with a
filter (optics) An optical filter is a device that selectively transmits light of different wavelengths, usually implemented as a glass plane or plastic device in the optical path, which are either dyed in the bulk or have interference coatings. The optical ...
photomultiplier A photomultiplier is a device that converts incident photons into an electrical signal. Kinds of photomultiplier include: * Photomultiplier tube, a vacuum tube converting incident photons into an electric signal. Photomultiplier tubes (PMTs for sh ...
system. * ''Spot'' or ''feature'': a small area on an array slide that contains picomoles of specific DNA samples. * For other relevant terms see: **
Glossary of gene expression terms A glossary (from grc, γλῶσσα, ''glossa''; language, speech, wording) also known as a vocabulary or clavis, is an alphabetical list of terms in a particular domain of knowledge with the definitions for those terms. Traditionally, a glos ...
**
Protocol (natural sciences) In natural and social science research, a protocol is most commonly a predefined procedural method in the design and implementation of an experiment. Protocols are written whenever it is desirable to standardize a laboratory method to ensure succe ...


See also

*
Transcriptomics technologies Transcriptomics technologies are the techniques used to study an organism's transcriptome, the sum of all of its RNA transcripts. The information content of an organism is recorded in the DNA of its genome and expressed through transcription. ...
**
Serial analysis of gene expression Serial Analysis of Gene Expression (SAGE) is a transcriptomic technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags that correspond to fragments of those tra ...
** RNA-Seq *
MAGIChip MAGIChips, also known as "microarrays of gel-immobilized compounds on a chip" or "three-dimensional DNA microarrays", are devices for molecular hybridization produced by immobilizing oligonucleotides, DNA, enzymes, antibodies, and other compounds o ...
*
Microarray analysis techniques Microarray analysis techniques are used in interpreting the data generated from experiments on DNA (Gene chip analysis), RNA, and protein microarrays, which allow researchers to investigate the expression state of a large number of genes - in many ...
*
Microarray databases A microarray database is a repository containing microarray gene expression data. The key uses of a microarray database are to store the measurement data, manage a searchable index, and make the data available to other applications for analysis and ...
*
Cyanine Cyanines, also referred to as tetramethylindo(di)-carbocyanines are a synthetic dye family belonging to the polymethine group. Although the name derives etymologically from terms for shades of blue, the cyanine family covers the electromagnetic ...
dyes, such as Cy3 and Cy5, are commonly used
fluorophores A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with sev ...
with microarrays *
Gene chip analysis In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
* Significance analysis of microarrays * Methylation specific oligonucleotide microarray *
Microfluidics Microfluidics refers to the behavior, precise control, and manipulation of fluids that are geometrically constrained to a small scale (typically sub-millimeter) at which surface forces dominate volumetric forces. It is a multidisciplinary field th ...
or lab-on-chip * Pathogenomics * Phenotype microarray * Systems biology *
Whole genome sequencing Whole genome sequencing (WGS), also known as full genome sequencing, complete genome sequencing, or entire genome sequencing, is the process of determining the entirety, or nearly the entirety, of the DNA sequence of an organism's genome at a ...


References


External links

* * * *
Microarray Animation
1Lec.com
PLoS Biology Primer: Microarray Analysis

Rundown of microarray technology

ArrayMining.net
free web-server for online microarray analysis
Microarray – How does it work?

PNAS Commentary: Discovery of Principles of Nature from Mathematical Modeling of DNA Microarray Data

DNA microarray virtual experiment
{{DEFAULTSORT:Dna Microarray Gene expression Bioinformatics Genetics techniques Glass coating and surface modification DNA Microtechnology Articles containing video clips Molecular biology techniques Microarrays