Minimizing off-target signals in RNA fluorescent in situ hybridization.
Identifieur interne : 001E30 ( PubMed/Checkpoint ); précédent : 001E29; suivant : 001E31Minimizing off-target signals in RNA fluorescent in situ hybridization.
Auteurs : Aaron Arvey [États-Unis] ; Anita Hermann ; Cheryl C. Hsia ; Eugene Ie ; Yoav Freund ; William McginnisSource :
- Nucleic acids research [ 1362-4962 ] ; 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 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.
DOI: 10.1093/nar/gkq042
PubMed: 20164092
Affiliations:
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pubmed:20164092Le document en format XML
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<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>
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