Minimizing off-target signals in RNA fluorescent in situ hybridization
Identifieur interne : 001355 ( Pmc/Checkpoint ); précédent : 001354; suivant : 001356Minimizing off-target signals in RNA fluorescent in situ hybridization
Auteurs : Aaron Arvey ; Anita Hermann [États-Unis] ; Cheryl C. Hsia [États-Unis] ; Eugene Ie [États-Unis] ; Yoav Freund ; William Mcginnis [États-Unis]Source :
- Nucleic Acids Research [ 0305-1048 ] ; 2010.
Abstract
Fluorescent
Url:
DOI: 10.1093/nar/gkq042
PubMed: 20164092
PubMed Central: 2879521
Affiliations:
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Hsia</name><affiliation wicri:level="2"><nlm:aff wicri:cut=" and" id="AFF1">Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Californie</region></placeName><wicri:cityArea>Division of Biological Sciences, University of California, San Diego, La Jolla</wicri:cityArea></affiliation></author><author><name sortKey="Ie, Eugene" sort="Ie, Eugene" uniqKey="Ie E" first="Eugene" last="Ie">Eugene Ie</name><affiliation><nlm:aff id="AFF1">Department of Computer Sciences and Engineering,</nlm:aff></affiliation><affiliation wicri:level="2"><nlm:aff id="AFF1">Google Inc., Mountain View, CA 94043, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Google Inc., Mountain View, CA 94043</wicri:regionArea><placeName><region type="state">Californie</region></placeName></affiliation></author><author><name sortKey="Freund, Yoav" sort="Freund, Yoav" uniqKey="Freund Y" first="Yoav" last="Freund">Yoav Freund</name><affiliation><nlm:aff id="AFF1">Department of Computer Sciences and Engineering,</nlm:aff></affiliation></author><author><name sortKey="Mcginnis, William" sort="Mcginnis, William" uniqKey="Mcginnis W" first="William" last="Mcginnis">William Mcginnis</name><affiliation wicri:level="2"><nlm:aff wicri:cut=" and" id="AFF1">Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Californie</region></placeName><wicri:cityArea>Division of Biological Sciences, University of California, San Diego, La Jolla</wicri:cityArea></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">20164092</idno><idno type="pmc">2879521</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2879521</idno><idno type="RBID">PMC:2879521</idno><idno type="doi">10.1093/nar/gkq042</idno><date when="2010">2010</date><idno type="wicri:Area/Pmc/Corpus">000F47</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000F47</idno><idno type="wicri:Area/Pmc/Curation">000F47</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000F47</idno><idno type="wicri:Area/Pmc/Checkpoint">001355</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Checkpoint">001355</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Minimizing off-target signals in RNA fluorescent <italic>in situ</italic> hybridization</title><author><name sortKey="Arvey, Aaron" sort="Arvey, Aaron" uniqKey="Arvey A" first="Aaron" last="Arvey">Aaron Arvey</name><affiliation><nlm:aff id="AFF1">Computational and Systems Biology Center, Memorial Sloan-Kettering Cancer Center, New York, NY, 10065,</nlm:aff><wicri:noCountry code="subfield">10065</wicri:noCountry></affiliation><affiliation><nlm:aff id="AFF1">Department of Computer Sciences and Engineering,</nlm:aff></affiliation></author><author><name sortKey="Hermann, Anita" sort="Hermann, Anita" uniqKey="Hermann A" first="Anita" last="Hermann">Anita Hermann</name><affiliation wicri:level="2"><nlm:aff wicri:cut=" and" id="AFF1">Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Californie</region></placeName><wicri:cityArea>Division of Biological Sciences, University of California, San Diego, La Jolla</wicri:cityArea></affiliation></author><author><name sortKey="Hsia, Cheryl C" sort="Hsia, Cheryl C" uniqKey="Hsia C" first="Cheryl C." last="Hsia">Cheryl C. Hsia</name><affiliation wicri:level="2"><nlm:aff wicri:cut=" and" id="AFF1">Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Californie</region></placeName><wicri:cityArea>Division of Biological Sciences, University of California, San Diego, La Jolla</wicri:cityArea></affiliation></author><author><name sortKey="Ie, Eugene" sort="Ie, Eugene" uniqKey="Ie E" first="Eugene" last="Ie">Eugene Ie</name><affiliation><nlm:aff id="AFF1">Department of Computer Sciences and Engineering,</nlm:aff></affiliation><affiliation wicri:level="2"><nlm:aff id="AFF1">Google Inc., Mountain View, CA 94043, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Google Inc., Mountain View, CA 94043</wicri:regionArea><placeName><region type="state">Californie</region></placeName></affiliation></author><author><name sortKey="Freund, Yoav" sort="Freund, Yoav" uniqKey="Freund Y" first="Yoav" last="Freund">Yoav Freund</name><affiliation><nlm:aff id="AFF1">Department of Computer Sciences and Engineering,</nlm:aff></affiliation></author><author><name sortKey="Mcginnis, William" sort="Mcginnis, William" uniqKey="Mcginnis W" first="William" last="Mcginnis">William Mcginnis</name><affiliation wicri:level="2"><nlm:aff wicri:cut=" and" id="AFF1">Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Californie</region></placeName><wicri:cityArea>Division of Biological Sciences, University of California, San Diego, La Jolla</wicri:cityArea></affiliation></author></analytic><series><title level="j">Nucleic Acids Research</title><idno type="ISSN">0305-1048</idno><idno type="eISSN">1362-4962</idno><imprint><date when="2010">2010</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Fluorescent <italic>in situ</italic> hybridization (FISH) techniques are becoming extremely sensitive, to the point where individual RNA or DNA molecules can be detected with small probes. At this level of sensitivity, the elimination of ‘off-target’ hybridization is of crucial importance, but typical probes used for RNA and DNA FISH contain sequences repeated elsewhere in the genome. We find that very short (e.g. 20 nt) perfect repeated sequences within much longer probes (e.g. 350–1500 nt) can produce significant off-target signals. The extent of noise is surprising given the long length of the probes and the short length of non-specific regions. When we removed the small regions of repeated sequence from either short or long probes, we find that the signal-to-noise ratio is increased by orders of magnitude, putting us in a regime where fluorescent signals can be considered to be a quantitative measure of target transcript numbers. As the majority of genes in complex organisms contain repeated <italic>k</italic>-mers, we provide genome-wide annotations of <italic>k</italic>-mer-uniqueness at <ext-link ext-link-type="uri" xlink:href="http://cbio.mskcc.org/~aarvey/repeatmap">http://cbio.mskcc.org/∼aarvey/repeatmap</ext-link>.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Kosman, D" uniqKey="Kosman D">D Kosman</name></author><author><name sortKey="Mizutani, C" uniqKey="Mizutani C">C Mizutani</name></author><author><name sortKey="Lemons, D" uniqKey="Lemons D">D Lemons</name></author><author><name sortKey="Cox, W" uniqKey="Cox W">W Cox</name></author><author><name sortKey="Mcginnis, W" uniqKey="Mcginnis W">W McGinnis</name></author><author><name sortKey="Bier, E" uniqKey="Bier E">E Bier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gregorieff, A" uniqKey="Gregorieff A">A Gregorieff</name></author><author><name 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uniqKey="Parthasarathy N">N Parthasarathy</name></author><author><name sortKey="Krause, H" uniqKey="Krause H">H Krause</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Nucleic Acids Res</journal-id><journal-id journal-id-type="iso-abbrev">Nucleic Acids Res</journal-id><journal-id journal-id-type="publisher-id">nar</journal-id><journal-id journal-id-type="hwp">nar</journal-id><journal-title-group><journal-title>Nucleic Acids Research</journal-title></journal-title-group><issn pub-type="ppub">0305-1048</issn><issn pub-type="epub">1362-4962</issn><publisher><publisher-name>Oxford University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">20164092</article-id><article-id pub-id-type="pmc">2879521</article-id><article-id pub-id-type="doi">10.1093/nar/gkq042</article-id><article-id pub-id-type="publisher-id">gkq042</article-id><article-categories><subj-group subj-group-type="heading"><subject>Methods Online</subject></subj-group></article-categories><title-group><article-title>Minimizing off-target signals in RNA fluorescent <italic>in situ</italic> hybridization</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Arvey</surname><given-names>Aaron</given-names></name><xref ref-type="aff" rid="AFF1"><sup>1</sup></xref><xref ref-type="aff" rid="AFF1"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Hermann</surname><given-names>Anita</given-names></name><xref ref-type="aff" rid="AFF1"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Hsia</surname><given-names>Cheryl C.</given-names></name><xref ref-type="aff" rid="AFF1"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Ie</surname><given-names>Eugene</given-names></name><xref ref-type="aff" rid="AFF1"><sup>2</sup></xref><xref ref-type="aff" rid="AFF1"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>Freund</surname><given-names>Yoav</given-names></name><xref ref-type="aff" rid="AFF1"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>McGinnis</surname><given-names>William</given-names></name><xref ref-type="aff" rid="AFF1"><sup>3</sup></xref><xref ref-type="corresp" rid="COR1">*</xref></contrib></contrib-group><aff id="AFF1"><sup>1</sup>Computational and Systems Biology Center, Memorial Sloan-Kettering Cancer Center, New York, NY, 10065,<sup>2</sup>Department of Computer Sciences and Engineering,<sup>3</sup>Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093 and<sup>4</sup>Google Inc., Mountain View, CA 94043, USA</aff><author-notes><corresp id="COR1">*To whom correspondence should be addressed. Tel: <phone>858 822 0461</phone>; Fax: <fax>858 822 3021</fax>; Email: <email>wmcginnis@ucsd.edu</email></corresp><fn><p>The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.</p></fn></author-notes><pub-date pub-type="ppub"><month>6</month><year>2010</year></pub-date><pub-date pub-type="epub"><day>17</day><month>2</month><year>2010</year></pub-date><pub-date pub-type="pmc-release"><day>17</day><month>2</month><year>2010</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on the
<volume>38</volume><issue>10</issue><fpage>e115</fpage><lpage>e115</lpage><history><date date-type="received"><day>4</day><month>11</month><year>2009</year></date><date date-type="rev-recd"><day>11</day><month>12</month><year>2009</year></date><date date-type="accepted"><day>17</day><month>1</month><year>2010</year></date></history><permissions><copyright-statement>© The Author(s) 2010. Published by Oxford University Press.</copyright-statement><copyright-year>2010</copyright-year><license license-type="creative-commons" xlink:href="http://creativecommons.org/licenses/by-nc/2.5"><license-p><pmc-comment>CREATIVE COMMONS</pmc-comment>This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc/2.5">http://creativecommons.org/licenses/by-nc/2.5</ext-link>), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><abstract><p>Fluorescent <italic>in situ</italic> hybridization (FISH) techniques are becoming extremely sensitive, to the point where individual RNA or DNA molecules can be detected with small probes. At this level of sensitivity, the elimination of ‘off-target’ hybridization is of crucial importance, but typical probes used for RNA and DNA FISH contain sequences repeated elsewhere in the genome. We find that very short (e.g. 20 nt) perfect repeated sequences within much longer probes (e.g. 350–1500 nt) can produce significant off-target signals. The extent of noise is surprising given the long length of the probes and the short length of non-specific regions. When we removed the small regions of repeated sequence from either short or long probes, we find that the signal-to-noise ratio is increased by orders of magnitude, putting us in a regime where fluorescent signals can be considered to be a quantitative measure of target transcript numbers. As the majority of genes in complex organisms contain repeated <italic>k</italic>-mers, we provide genome-wide annotations of <italic>k</italic>-mer-uniqueness at <ext-link ext-link-type="uri" xlink:href="http://cbio.mskcc.org/~aarvey/repeatmap">http://cbio.mskcc.org/∼aarvey/repeatmap</ext-link>.</p></abstract></article-meta></front></pmc><affiliations><list><country><li>États-Unis</li></country><region><li>Californie</li></region></list><tree><noCountry><name sortKey="Arvey, Aaron" sort="Arvey, Aaron" uniqKey="Arvey A" first="Aaron" last="Arvey">Aaron Arvey</name><name sortKey="Freund, Yoav" sort="Freund, Yoav" uniqKey="Freund Y" first="Yoav" last="Freund">Yoav Freund</name></noCountry><country name="États-Unis"><region name="Californie"><name sortKey="Hermann, Anita" sort="Hermann, Anita" uniqKey="Hermann A" first="Anita" last="Hermann">Anita Hermann</name></region><name sortKey="Hsia, Cheryl C" sort="Hsia, Cheryl C" uniqKey="Hsia C" first="Cheryl C." last="Hsia">Cheryl C. Hsia</name><name sortKey="Ie, Eugene" sort="Ie, Eugene" uniqKey="Ie E" first="Eugene" last="Ie">Eugene Ie</name><name sortKey="Mcginnis, William" sort="Mcginnis, William" uniqKey="Mcginnis W" first="William" last="Mcginnis">William Mcginnis</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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