Editing DNA microarray (section)

== Uses and types ==
[[Image:Affymetrix-microarray.jpg|thumb|right|150px|Two Affymetrix chips. A [[match]] 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 [[gene expression|expression analysis]] or [[expression profiling]].

Applications include:

{| class="wikitable"
|-
! Application or technology
! Synopsis
|-
| [[Gene expression profiling]]
| In an [[mRNA]] or [[gene expression profiling]] experiment the [[Gene expression|expression]] levels of thousands of genes are simultaneously monitored to study the effects of certain treatments, [[disease]]s, and developmental stages on gene expression. For example, microarray-based gene expression profiling can be used to identify genes whose expression is changed in response to [[pathogens]] or other organisms by comparing gene expression in infected to that in uninfected cells or tissues.<ref name="Adomas et al.">{{cite journal|author=Adomas A|author2=Heller G|author3=Olson A|author4=Osborne J|author5=Karlsson M|author6=Nahalkova J|author7=Van Zyl L|author8=Sederoff R|author9=Stenlid J|author10=Finlay R|author11=Asiegbu FO|date=2008|title=Comparative analysis of transcript abundance in Pinus sylvestris after challenge with a saprotrophic, pathogenic or mutualistic fungus|journal=Tree Physiol.|volume=28|pages=885–897|pmid=18381269|issue=6|doi=10.1093/treephys/28.6.885|doi-access=}}</ref>
|-
| [[Comparative genomic hybridization]]
| Assessing genome content in different cells or closely related organisms, as originally described by [[Patrick O. Brown|Patrick Brown]], Jonathan Pollack, [[Ash Alizadeh]] and colleagues at [[Stanford University|Stanford]].<ref name="Pollack et al.">{{cite journal|author=Pollack JR|author2=Perou CM|author3=Alizadeh AA|author4=Eisen MB|author5=Pergamenschikov A|author6=Williams CF|author7=Jeffrey SS|author8=Botstein D|author9=Brown PO|date= 1999|title=Genome-wide analysis of DNA copy-number changes using cDNA microarrays|journal=Nat Genet|volume=23|pages=41–46|pmid=10471496|doi=10.1038/12640|issue=1|s2cid=997032|url=https://cdr.lib.unc.edu/downloads/sj139421j }}</ref><ref name="Moran et al.">{{cite journal|author=Moran G|author2=Stokes C|author3=Thewes S|author4=Hube B|author5=Coleman DC|author6=Sullivan D|date= 2004|title=Comparative genomics using Candida albicans DNA microarrays reveals absence and divergence of virulence-associated genes in Candida dubliniensis|journal=Microbiology|volume=150|pages=3363–3382|pmid=15470115|doi=10.1099/mic.0.27221-0|issue=Pt 10|doi-access=free|hdl=2262/6097|hdl-access=free}}</ref>
|-
| GeneID
| Small microarrays to check IDs of organisms in food and feed (like [[GMO]] [https://web.archive.org/web/20090228210111/http://bgmo.jrc.ec.europa.eu/home/docs.htm]), [[mycoplasms]] in cell culture, or [[pathogens]] for disease detection, mostly combining [[Polymerase chain reaction|PCR]] and microarray technology.
|-
| [[ChIP-on-chip|Chromatin immunoprecipitation on Chip]]
| DNA sequences bound to a particular protein can be isolated by [[immunoprecipitation|immunoprecipitating]] that protein ([[Chromatin immunoprecipitation|ChIP]]), these fragments can be then hybridized to a microarray (such as a [[tiling array]]) allowing the determination of protein binding site occupancy throughout the genome. Example protein to [[Chromatin immunoprecipitation|immunoprecipitate]] are histone modifications ([[H3K27me3]], H3K4me2, H3K9me3, etc.), [[Polycomb-group protein]] (PRC2:Suz12, PRC1:YY1) and [[trithorax-group protein]] (Ash1) to study the [[epigenetics|epigenetic landscape]] or [[RNA polymerase II]] to study the [[Transcription (genetics)|transcription landscape]].
|-
| [[DNA adenine methyltransferase identification|DamID]]
| Analogously to [[ChIP]], genomic regions bound by a protein of interest can be isolated and used to probe a microarray to determine binding site occupancy. Unlike ChIP, DamID does not require antibodies but makes use of adenine methylation near the protein's binding sites to selectively amplify those regions, introduced by expressing minute amounts of protein of interest fused to bacterial [[Dam (methylase)|DNA adenine methyltransferase]].
|-
| [[SNP array|SNP detection]]
| Identifying [[single nucleotide polymorphism]] among [[alleles]] within or between populations.<ref name="Hacia et al.">{{cite journal |author=Hacia JG|author2=Fan JB|author3=Ryder O|author4= Jin L|author5=Edgemon K|author6=Ghandour G|author7=Mayer RA|author8= Sun B|author9=Hsie L|author10=Robbins CM|author11=Brody LC|author12=Wang D|author13=Lander ES|author14=Lipshutz R|author15=Fodor SP|author16=Collins FS|date= 1999|title=Determination of ancestral alleles for human single-nucleotide polymorphisms using high-density oligonucleotide arrays|journal=Nat Genet|volume=22|pages=164–167|pmid=10369258 | doi = 10.1038/9674|issue=2|s2cid=41718227}}</ref> Several applications of microarrays make use of SNP detection, including [[genotyping]], [[forensic]] analysis, measuring [[Genetic predisposition|predisposition]] to disease, identifying drug-candidates, evaluating [[germline]] mutations in individuals or [[Somatic (biology)|somatic]] mutations in cancers, assessing [[loss of heterozygosity]], or [[genetic linkage]] analysis.
|-
| [[Alternative splicing]] detection
| An ''[[exon junction array]]'' design uses probes specific to the expected or potential splice sites of predicted [[exon]]s for a gene. It is of intermediate density, or coverage, to a typical gene expression array (with 1–3 probes per gene) and a genomic tiling array (with hundreds or thousands of probes per gene). It is used to assay the expression of alternative splice forms of a gene. [[Exon array]]s have a different design, employing probes designed to detect each individual exon for known or predicted genes, and can be used for detecting different splicing isoforms.
|-
| [[Fusion gene]]s microarray
| A fusion gene microarray can detect fusion transcripts, ''e.g.'' from cancer specimens. The principle behind this is building on the [[alternative splicing]] microarrays. The oligo design strategy enables combined measurements of chimeric transcript junctions with exon-wise measurements of individual fusion partners.
|-
| [[Tiling array]]
| Genome tiling arrays consist of overlapping probes designed to densely represent a genomic region of interest, sometimes as large as an entire human chromosome. The purpose is to empirically detect expression of [[mRNA|transcripts]] or [[Alternative splicing|alternatively spliced forms]] which may not have been previously known or predicted.
|-
|Double-stranded B-DNA microarrays
|Right-handed double-stranded B-DNA microarrays can be used to characterize novel drugs and biologicals that can be employed to bind specific regions of immobilized, intact, double-stranded DNA. This approach can be used to inhibit gene expression.<ref name="Gagna 895–914">{{Cite journal|title = Novel multistranded, alternative, plasmid and helical transitional DNA and RNA microarrays: implications for therapeutics|journal = Pharmacogenomics|date = 2009-05-01|issn = 1744-8042|pmid = 19450135|pages = 895–914|volume = 10|issue = 5|doi = 10.2217/pgs.09.27|first1 = Claude E.|last1 = Gagna|first2 = W. Clark|last2 = Lambert}}</ref><ref name="Gagna 381–401">{{Cite journal|title = Cell biology, chemogenomics and chemoproteomics – application to drug discovery|journal = Expert Opinion on Drug Discovery|date = 2007-03-01|issn = 1746-0441|pmid = 23484648|pages = 381–401|volume = 2|issue = 3|doi = 10.1517/17460441.2.3.381|first1 = Claude E.|last1 = Gagna|first2 = W.|last2 = Clark Lambert|s2cid = 41959328}}</ref> They also allow for characterization of their structure under different environmental conditions.
|-
|Double-stranded Z-DNA microarrays
|Left-handed double-stranded Z-DNA microarrays can be used to identify short sequences of the alternative Z-DNA structure located within longer stretches of right-handed B-DNA genes (e.g., transcriptional enhancement, recombination, RNA editing).<ref name="Gagna 895–914"/><ref name="Gagna 381–401"/> The microarrays also allow for characterization of their structure under different environmental conditions.
|-
|Multi-stranded DNA microarrays (triplex-DNA microarrays and quadruplex-DNA microarrays)
|Multi-stranded DNA and RNA microarrays can be used to identify novel drugs that bind to these multi-stranded nucleic acid sequences. This approach can be used to discover new drugs and biologicals that have the ability to inhibit gene expression.<ref name="Gagna 895–914"/><ref name="Gagna 381–401"/><ref>{{Cite journal|title = Triplex technology in studies of DNA damage, DNA repair, and mutagenesis|journal = Biochimie|date = 2011-08-01|issn = 1638-6183|pmc = 3545518|pmid = 21501652|pages = 1197–1208|volume = 93|issue = 8|doi = 10.1016/j.biochi.2011.04.001|first1 = Anirban|last1 = Mukherjee|first2 = Karen M.|last2 = Vasquez}}</ref><ref>{{Cite journal|title = G-quadruplexes and their regulatory roles in biology|journal = Nucleic Acids Research|date = 2015-10-15|issn = 1362-4962|pmc = 4605312|pmid = 26350216|pages = 8627–8637|volume = 43|issue = 18|doi = 10.1093/nar/gkv862|first1 = Daniela|last1 = Rhodes|first2 = Hans J.|last2 = Lipps}}</ref> These microarrays also allow for characterization of their structure under different environmental conditions.
|}

Specialised arrays tailored to particular [[crop]]s are becoming increasingly popular in [[molecular breeding]] applications. In the future they could be used to screen [[seedling]]s at early stages to lower the number of unneeded seedlings tried out in breeding operations.<ref name="Rasheed-et-al-2017">{{cite journal | last1=Rasheed | first1=Awais | last2=Hao | first2=Yuanfeng | last3=Xia | first3=Xianchun | last4=Khan | first4=Awais | last5=Xu | first5=Yunbi | last6=Varshney | first6=Rajeev K. | last7=He | first7=Zhonghu | title=Crop Breeding Chips and Genotyping Platforms: Progress, Challenges, and Perspectives | journal=[[Molecular Plant]] | publisher=[[Chinese Academy of Sciences|Chin Acad Sci]]+[[Chinese Society for Plant Biology|Chin Soc Plant Bio]]+[[Shanghai Institutes for Biological Sciences|Shanghai Inst Bio Sci]] ([[Elsevier]]) | volume=10 | issue=8 | year=2017 | issn=1674-2052 | doi=10.1016/j.molp.2017.06.008 | pages=1047–1064 | s2cid=33780984 | pmid=28669791| doi-access=free | bibcode=2017MPlan..10.1047R | url=http://oar.icrisat.org/10133/1/S1674-2052%2817%2930174-0.pdf }}</ref>

=== 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.
<!-- Repetition of second paragraph but less detail
=== Surface engineering ===

The first step of DNA microarray fabrication involves [[surface engineering]] of a substrate in order to obtain desirable surface properties for the application of interest. Optimal surface properties are those which produce high signal to noise ratios for the DNA targets of interest. Generally, this involves maximizing the probe surface density and activity while minimizing the non-specific binding of the targets of interest.{{Citation needed|date=October 2008}} Methods of surface engineering vary depending on the platform material, design, and application.{{Citation needed|date=October 2008}} However micro-array system can be  modified.
-->

=== Spotted vs. ''in situ'' synthesised arrays ===
[[File:Microarray printing.ogv|thumb|A DNA microarray being printed by a [[robot]] 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,<ref>J Biochem Biophys Methods. 2000 Mar 16;42(3):105–10. DNA-printing: utilization of a standard inkjet printer for the transfer of nucleic acids to solid supports. Goldmann T, Gonzalez JS.</ref><ref>{{cite journal|journal=Genome Biology | title=POSaM: a fast, flexible, open-source, inkjet oligonucleotide synthesizer and microarrayer| author=Lausted C| volume = 5 | pages=R58 | doi=10.1186/gb-2004-5-8-r58 | pmid=15287980 | date=2004| issue=8| pmc=507883|display-authors=etal| doi-access=free}}</ref> or [[electrochemistry]] on microelectrode arrays.

In ''spotted microarrays'', the probes are [[oligonucleotide synthesis|oligonucleotide]]s, [[cDNA]] or small fragments of [[Polymerase chain reaction|PCR]] products that correspond to [[mRNA]]s. The probes are [[oligonucleotide synthesis|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,<ref name="TRC Standardization">{{cite journal |date=2005 |title=Standardizing global gene expression analysis between laboratories and across platforms |journal=Nat Methods |volume=2 |pages=351–356 |pmid=15846362 |doi=10.1038/nmeth754 |last12=Deng |first12=S |last13=Dressman |first13=HK |last14=Fannin |first14=RD |last15=Farin |first15=FM |last16=Freedman |first16=JH |last17=Fry |first17=RC |last18=Harper |first18=A |last19=Humble |first19=MC |last20=Hurban |first20=P |last21=Kavanagh |first21=TJ |last22=Kaufmann |first22=WK |first23=KF |first24=L |first25=JA |first26=MR |last27=Li |first27=J |first28=YJ |last29=Lobenhofer |first29=EK |last30=Lu |last31=Malek |first31=RL |last32=Milton |first32=S |last33=Nagalla |first33=SR |last34=O'malley |first34=JP |last35=Palmer |first35=VS |last36=Pattee |first36=P |last7=Paules |first7=RS |last38=Perou |first38=CM |last9=Phillips |first39=K |last40=Qin |last41=Qiu |first41=Y |last42=Quigley |first42=SD |last43=Rodland |first43=M |last44=Rusyn |first44=I |last45=Samson |first45= LD|last46= Schwartz|last47=Shi |first47=Y |last48=Shin |last49=Sieber |last50=Slifer |last51=Speer |first51=MC |last52=Spencer |first52=PS |last53=Sproles |first53=DI |last54=Swenberg |first54=JA |last55=Suk|first55= WA |last56=Sullivan |first56=RC |last57=Tian |first57=R |last58=Tennant |first58=RW |last59= Todd |first59=SA |last60=Tucker |first60=CJ |last61=Van Houten |first61=B |last62=Weis |first62=BK |last63=Xuan |first63=S |last64=Zarbl |first64=H |last65=Members of the Toxicogenomics Research |first65=Consortium |issue=5 |author1=Bammler T, Beyer RP |author2=Consortium, Members of the Toxicogenomics Research |last3=Kerr |last4=Jing |last5=Lapidus |last6=Lasarev |last8=Li |first3=X |first4=LX |first6=DA |first8=JL |first9=SO |first5=S |s2cid=195368323 }}</ref> 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]]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 [[oligonucleotide synthesis|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.<ref name="Affy PNAS Paper">{{cite journal|author=Pease AC|author2=Solas D|author3=Sullivan EJ|author4=Cronin MT|author5=Holmes CP|author6=Fodor SP|date= 1994|title=Light-generated oligonucleotide arrays for rapid DNA sequence analysis|journal=PNAS|volume=91|pages=5022–5026|pmid=8197176|doi=10.1073/pnas.91.11.5022|issue=11|pmc=43922|bibcode=1994PNAS...91.5022P|doi-access=free}}</ref> 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.<ref name="NimbleGen Genome Res Paper">{{cite journal|author=Nuwaysir EF|author2=Huang W|author3=Albert TJ|author4=Singh J|author5=Nuwaysir K|author6=Pitas A|author7=Richmond T|author8=Gorski T|author9=Berg JP|author10=Ballin J|author11=McCormick M|author12=Norton J|author13=Pollock T|author14=Sumwalt T|author15=Butcher L|author16=Porter D|author17=Molla M|author18=Hall C|author19=Blattner F|author20=Sussman MR|author21=Wallace RL|author22=Cerrina F|author23=Green RD|date= 2002|title=Gene Expression Analysis Using Oligonucleotide Arrays Produced by Maskless Photolithography|journal=Genome Res|volume=12|pages=1749–1755|pmid=12421762|doi=10.1101/gr.362402|issue=11|pmc=187555}}</ref>

=== Two-channel vs. one-channel detection ===
[[Image:Microarray-schema.jpg|thumb|right|Diagram of typical dual-colour [[DNA microarray experiment|microarray experiment]] ]]
<!--- channel is the correct word and colour is a bit wrong semantically, see discussion --->
''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&nbsp;nm (corresponding to the green part of the light spectrum), and [[Cyanine|Cy]]5 with a fluorescence emission wavelength of 670&nbsp;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 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 <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.  
{| class="wikitable"
!number of samples
!one-channel microarray
!two channel microarray
!
two channel microarray (with reference)
|-
|1
|1
|1
|1
|-
|2
|2
|1
|1
|-
|3
|3
|3
|2
|-
|4
|4
|6
|3
|-
|<math>i</math>
|<math>i</math>
|<math>i(i-1)/2</math>
|<math>i -1</math>
|}

===A typical protocol===
[[File:Summary of RNA Microarray.svg|thumb|''Examples of levels of application of microarrays.'' Within the organisms, genes are transcribed and spliced to produce mature mRNA transcripts (red). The mRNA is extracted from the organism and reverse transcriptase is used to copy the mRNA into stable ds-cDNA (blue). In microarrays, the ds-cDNA is fragmented and fluorescently labelled (orange). The labelled fragments bind to an ordered array of complementary oligonucleotides, and [[Fluorometer|measurement of fluorescent intensity]] 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.<ref>{{Cite journal|last1=Shafee|first1=Thomas|last2=Lowe|first2=Rohan|date=2017|title=Eukaryotic and prokaryotic gene structure|journal=WikiJournal of Medicine|language=en|volume=4|issue=1|doi=10.15347/wjm/2017.002|issn=2002-4436|doi-access=free|s2cid=35766676 }}</ref>]]

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-control|case]]) and untreated sample ([[Case-control|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 [[Chromatin immunoprecipitation|immunoprecipitated]] ([[ChIP-on-chip]]) for [[Epigenetics|epigenetic]] or regulation studies. In this example total RNA is isolated (both nuclear and [[cytoplasm]]ic) 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 [[Polymerase chain reaction|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]]). [[MicroRNA|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 (molecular biology)|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 [[radioactivity in biology|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 [[N-hydroxysuccinimide|NHS]] amino-reactive dyes (such as [[cyanine|cyanine dyes]]); for single-channel arrays, the coupling stage occurs after hybridization, using [[Biotin#Use in biotechnology|biotin]] and labeled [[Streptavidin#Uses in biotechnology|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 [[DNA separation by silica adsorption|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 [[Artifact (error)|artifact]]s 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 [[nucleic acid hybridization|hybridization]] solution which can consist of [[Sodium dodecyl sulfate|SDS]], [[citrate|SSC]], [[dextran|dextran sulfate]], a blocking agent (such as [[Comparative genomic hybridization#Blocking|Cot-1 DNA]], salmon sperm DNA, calf thymus DNA, [[PolyA]], or PolyT), [[Denhardt's solution]], or [[Methylamine|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-score|z-ratio]], [[local regression|loess and lowess regression]] and RMA (robust multichip analysis) for Affymetrix chips (single-channel, silicon chip, ''in situ'' synthesized short oligonucleotides).