Minimizing off-target signals in RNA fluorescent in situ hybridization
Identifieur interne : 000737 ( Ncbi/Merge ); précédent : 000736; suivant : 000738Minimizing 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.
Descripteurs français
- KwdFr :
- ARN messager (), ARN messager (analyse), Animaux, Drosophila melanogaster (embryologie), Drosophila melanogaster (génétique), Embryon non mammalien (), Facteurs de transcription (génétique), Protéines de Drosophila (génétique), Protéines nucléaires (génétique), Sondes d'ARN (), Séquences répétées d'acides nucléiques, Technique FISH ().
- MESH :
- analyse : ARN messager.
- embryologie : Drosophila melanogaster.
- génétique : Drosophila melanogaster, Facteurs de transcription, Protéines de Drosophila, Protéines nucléaires.
- ARN messager, Animaux, Embryon non mammalien, Sondes d'ARN, Séquences répétées d'acides nucléiques, Technique FISH.
English descriptors
- KwdEn :
- Animals, Drosophila Proteins (genetics), Drosophila melanogaster (embryology), Drosophila melanogaster (genetics), Embryo, Nonmammalian (chemistry), In Situ Hybridization, Fluorescence (methods), Nuclear Proteins (genetics), RNA Probes (chemistry), RNA, Messenger (analysis), RNA, Messenger (chemistry), Repetitive Sequences, Nucleic Acid, Transcription Factors (genetics).
- MESH :
- chemical , analysis : RNA, Messenger.
- chemical , chemistry : RNA Probes, RNA, Messenger.
- chemical , genetics : Drosophila Proteins, Nuclear Proteins, Transcription Factors.
- chemistry : Embryo, Nonmammalian.
- embryology : Drosophila melanogaster.
- genetics : Drosophila melanogaster.
- methods : In Situ Hybridization, Fluorescence.
- Animals, Repetitive Sequences, Nucleic Acid.
Abstract
Fluorescent
Url:
DOI: 10.1093/nar/gkq042
PubMed: 20164092
PubMed Central: 2879521
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Links to Exploration step
PMC:2879521Le document en format XML
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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></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><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Drosophila Proteins (genetics)</term><term>Drosophila melanogaster (embryology)</term><term>Drosophila melanogaster (genetics)</term><term>Embryo, Nonmammalian (chemistry)</term><term>In Situ Hybridization, Fluorescence (methods)</term><term>Nuclear Proteins (genetics)</term><term>RNA Probes (chemistry)</term><term>RNA, Messenger (analysis)</term><term>RNA, Messenger (chemistry)</term><term>Repetitive Sequences, Nucleic Acid</term><term>Transcription Factors (genetics)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ARN messager ()</term><term>ARN messager (analyse)</term><term>Animaux</term><term>Drosophila melanogaster (embryologie)</term><term>Drosophila melanogaster (génétique)</term><term>Embryon non mammalien ()</term><term>Facteurs de transcription (génétique)</term><term>Protéines de Drosophila (génétique)</term><term>Protéines nucléaires (génétique)</term><term>Sondes d'ARN ()</term><term>Séquences répétées d'acides nucléiques</term><term>Technique FISH ()</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analysis" xml:lang="en"><term>RNA, Messenger</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>RNA Probes</term><term>RNA, Messenger</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Drosophila Proteins</term><term>Nuclear Proteins</term><term>Transcription Factors</term></keywords><keywords scheme="MESH" qualifier="analyse" xml:lang="fr"><term>ARN messager</term></keywords><keywords scheme="MESH" qualifier="chemistry" xml:lang="en"><term>Embryo, Nonmammalian</term></keywords><keywords scheme="MESH" qualifier="embryologie" xml:lang="fr"><term>Drosophila melanogaster</term></keywords><keywords scheme="MESH" qualifier="embryology" xml:lang="en"><term>Drosophila melanogaster</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Drosophila melanogaster</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Drosophila melanogaster</term><term>Facteurs de transcription</term><term>Protéines de Drosophila</term><term>Protéines nucléaires</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>In Situ Hybridization, Fluorescence</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Repetitive Sequences, Nucleic Acid</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>ARN messager</term><term>Animaux</term><term>Embryon non mammalien</term><term>Sondes d'ARN</term><term>Séquences répétées d'acides nucléiques</term><term>Technique FISH</term></keywords></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="Sanchez Herrero E">E Sánchez-Herrero</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Angerer, L" uniqKey="Angerer L">L Angerer</name></author><author><name sortKey="Angerer, R" uniqKey="Angerer R">R Angerer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Brahic, M" uniqKey="Brahic M">M Brahic</name></author><author><name sortKey="Haase, A" uniqKey="Haase A">A Haase</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cox, K" uniqKey="Cox K">K Cox</name></author><author><name sortKey="Deleon, D" uniqKey="Deleon D">D DeLeon</name></author><author><name sortKey="Angerer, L" uniqKey="Angerer L">L Angerer</name></author><author><name sortKey="Angerer, R" uniqKey="Angerer R">R Angerer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lecuyer, E" uniqKey="Lecuyer E">E Lecuyer</name></author><author><name sortKey="Parthasarathy, N" 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><double pmid="20164092"><pmc><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">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></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><pubmed><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Minimizing off-target signals in RNA fluorescent in situ hybridization.</title><author><name sortKey="Arvey, Aaron" sort="Arvey, Aaron" uniqKey="Arvey A" first="Aaron" last="Arvey">Aaron Arvey</name><affiliation wicri:level="2"><nlm:affiliation>Computational and Systems Biology Center, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Computational and Systems Biology Center, Memorial Sloan-Kettering Cancer Center, New York, NY 10065</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation></author><author><name sortKey="Hermann, Anita" sort="Hermann, Anita" uniqKey="Hermann A" first="Anita" last="Hermann">Anita Hermann</name></author><author><name sortKey="Hsia, Cheryl C" sort="Hsia, Cheryl C" uniqKey="Hsia C" first="Cheryl C" last="Hsia">Cheryl C. Hsia</name></author><author><name sortKey="Ie, Eugene" sort="Ie, Eugene" uniqKey="Ie E" first="Eugene" last="Ie">Eugene Ie</name></author><author><name sortKey="Freund, Yoav" sort="Freund, Yoav" uniqKey="Freund Y" first="Yoav" last="Freund">Yoav Freund</name></author><author><name sortKey="Mcginnis, William" sort="Mcginnis, William" uniqKey="Mcginnis W" first="William" last="Mcginnis">William Mcginnis</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2010">2010</date><idno type="RBID">pubmed:20164092</idno><idno type="pmid">20164092</idno><idno type="doi">10.1093/nar/gkq042</idno><idno type="wicri:Area/PubMed/Corpus">001F66</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">001F66</idno><idno type="wicri:Area/PubMed/Curation">001F66</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">001F66</idno><idno type="wicri:Area/PubMed/Checkpoint">001E30</idno><idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">001E30</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Minimizing off-target signals in RNA fluorescent in situ hybridization.</title><author><name sortKey="Arvey, Aaron" sort="Arvey, Aaron" uniqKey="Arvey A" first="Aaron" last="Arvey">Aaron Arvey</name><affiliation wicri:level="2"><nlm:affiliation>Computational and Systems Biology Center, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Computational and Systems Biology Center, Memorial Sloan-Kettering Cancer Center, New York, NY 10065</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation></author><author><name sortKey="Hermann, Anita" sort="Hermann, Anita" uniqKey="Hermann A" first="Anita" last="Hermann">Anita Hermann</name></author><author><name sortKey="Hsia, Cheryl C" sort="Hsia, Cheryl C" uniqKey="Hsia C" first="Cheryl C" last="Hsia">Cheryl C. Hsia</name></author><author><name sortKey="Ie, Eugene" sort="Ie, Eugene" uniqKey="Ie E" first="Eugene" last="Ie">Eugene Ie</name></author><author><name sortKey="Freund, Yoav" sort="Freund, Yoav" uniqKey="Freund Y" first="Yoav" last="Freund">Yoav Freund</name></author><author><name sortKey="Mcginnis, William" sort="Mcginnis, William" uniqKey="Mcginnis W" first="William" last="Mcginnis">William Mcginnis</name></author></analytic><series><title level="j">Nucleic acids research</title><idno type="eISSN">1362-4962</idno><imprint><date when="2010" type="published">2010</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Drosophila Proteins (genetics)</term><term>Drosophila melanogaster (embryology)</term><term>Drosophila melanogaster (genetics)</term><term>Embryo, Nonmammalian (chemistry)</term><term>In Situ Hybridization, Fluorescence (methods)</term><term>Nuclear Proteins (genetics)</term><term>RNA Probes (chemistry)</term><term>RNA, Messenger (analysis)</term><term>RNA, Messenger (chemistry)</term><term>Repetitive Sequences, Nucleic Acid</term><term>Transcription Factors (genetics)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ARN messager ()</term><term>ARN messager (analyse)</term><term>Animaux</term><term>Drosophila melanogaster (embryologie)</term><term>Drosophila melanogaster (génétique)</term><term>Embryon non mammalien ()</term><term>Facteurs de transcription (génétique)</term><term>Protéines de Drosophila (génétique)</term><term>Protéines nucléaires (génétique)</term><term>Sondes d'ARN ()</term><term>Séquences répétées d'acides nucléiques</term><term>Technique FISH ()</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analysis" xml:lang="en"><term>RNA, Messenger</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>RNA Probes</term><term>RNA, Messenger</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Drosophila Proteins</term><term>Nuclear Proteins</term><term>Transcription Factors</term></keywords><keywords scheme="MESH" qualifier="analyse" xml:lang="fr"><term>ARN messager</term></keywords><keywords scheme="MESH" qualifier="chemistry" xml:lang="en"><term>Embryo, Nonmammalian</term></keywords><keywords scheme="MESH" qualifier="embryologie" xml:lang="fr"><term>Drosophila melanogaster</term></keywords><keywords scheme="MESH" qualifier="embryology" xml:lang="en"><term>Drosophila melanogaster</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Drosophila melanogaster</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Drosophila melanogaster</term><term>Facteurs de transcription</term><term>Protéines de Drosophila</term><term>Protéines nucléaires</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>In Situ Hybridization, Fluorescence</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Repetitive Sequences, Nucleic Acid</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>ARN messager</term><term>Animaux</term><term>Embryon non mammalien</term><term>Sondes d'ARN</term><term>Séquences répétées d'acides nucléiques</term><term>Technique FISH</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Fluorescent in situ 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 k-mers, we provide genome-wide annotations of k-mer-uniqueness at http://cbio.mskcc.org/ approximately aarvey/repeatmap.</div></front></TEI></pubmed></double></record>Pour manipuler ce document sous Unix (Dilib)
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