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{{short description|Collection of microscopic DNA spots attached to a solid surface}}
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[[File:From_spit_to_DNA-sample.webm|thumb|''How to use a microarray for genotyping.'' The video shows the process of extracting genotypes from a human spit sample using microarrays. Genotyping is a major use of DNA microarrays, but with some modifications they can also be used for other purposes such as measurement of gene expression and epigenetic markers.]]
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[[File:NA hybrid.svg|thumb|Hybridization of the target to the probe]]
{{Main|Nucleic acid hybridization}}
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The core principle behind microarrays is hybridization between two DNA strands, the property of [[Complementarity (molecular biology)|complementary]] nucleic acid sequences to specifically pair with each other by forming [[hydrogen bond]]s between complementary [[Nucleotide|nucleotide base pairs]]. A high number of complementary base pairs in a nucleotide sequence means tighter [[non-covalent]] 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, <!-- 2 channel experiments are mentioned below! so do not repeat --> and the identity of the feature is known by its position.
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| 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.
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| [[ChIP-on-chip|Chromatin immunoprecipitation on Chip]]
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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}}</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.
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# 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]].
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