Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization
We present a protocol for measuring the absolute number of mRNA molecules from a gene of interest in individual, chemically fixed Escherichia coli cells. A set of fluorescently labeled oligonucleotide probes is hybridized to the target mRNA, such that each mRNA molecule is decorated by a known numbe...
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| Veröffentlicht in: | Nature protocols 2013-06, Vol.8 (6), p.1100-1113 |
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| creator | Skinner, Samuel O Sepúlveda, Leonardo A Xu, Heng Golding, Ido |
| description | We present a protocol for measuring the absolute number of mRNA molecules from a gene of interest in individual, chemically fixed
Escherichia coli
cells. A set of fluorescently labeled oligonucleotide probes is hybridized to the target mRNA, such that each mRNA molecule is decorated by a known number of fluorescent dyes. Cells are then imaged using fluorescence microscopy. The copy number of the target mRNA is estimated from the total intensity of fluorescent foci in the cell, rather than from counting discrete 'spots' as in other currently available protocols. Image analysis is performed using an automated algorithm. The measured mRNA copy number distribution obtained from many individual cells can be used to extract the parameters of stochastic gene activity, namely the frequency and size of transcription bursts from the gene of interest. The experimental procedure takes 2 d, with another 2–3 d typically required for image and data analysis. |
| doi_str_mv | 10.1038/nprot.2013.066 |
| format | Article |
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Escherichia coli
cells. A set of fluorescently labeled oligonucleotide probes is hybridized to the target mRNA, such that each mRNA molecule is decorated by a known number of fluorescent dyes. Cells are then imaged using fluorescence microscopy. The copy number of the target mRNA is estimated from the total intensity of fluorescent foci in the cell, rather than from counting discrete 'spots' as in other currently available protocols. Image analysis is performed using an automated algorithm. The measured mRNA copy number distribution obtained from many individual cells can be used to extract the parameters of stochastic gene activity, namely the frequency and size of transcription bursts from the gene of interest. The experimental procedure takes 2 d, with another 2–3 d typically required for image and data analysis.</description><identifier>ISSN: 1754-2189</identifier><identifier>EISSN: 1750-2799</identifier><identifier>DOI: 10.1038/nprot.2013.066</identifier><identifier>PMID: 23680982</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>631/1647/2017/1947 ; 631/1647/2217/457/649/2157 ; 631/1647/245/2225 ; Algorithms ; Analytical Chemistry ; Automation ; Biological Techniques ; Cells ; Computational Biology/Bioinformatics ; Copy number ; Data analysis ; Dyes ; E coli ; Escherichia coli ; Escherichia coli - genetics ; Fluorescence ; Fluorescence in situ hybridization ; Fluorescence microscopy ; Fluorescent dyes ; Fluorescent indicators ; Genetic aspects ; Genetic variation ; Hybridization ; Image analysis ; Image processing ; In situ hybridization ; In Situ Hybridization, Fluorescence - methods ; Life Sciences ; Measurement ; Microarrays ; Microscopy, Fluorescence ; Oligonucleotide Probes - genetics ; Oligonucleotides ; Organic Chemistry ; Physiological aspects ; Probes ; Protocol ; RNA, Messenger - analysis ; Stochasticity ; Transcription</subject><ispartof>Nature protocols, 2013-06, Vol.8 (6), p.1100-1113</ispartof><rights>Springer Nature Limited 2013</rights><rights>COPYRIGHT 2013 Nature Publishing Group</rights><rights>Copyright Nature Publishing Group Jun 2013</rights><rights>Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. 2013.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c689t-2ec650b6d01cb60812358b1437357a936f4b7e9aac36547cda8f974f6d2484c03</citedby><cites>FETCH-LOGICAL-c689t-2ec650b6d01cb60812358b1437357a936f4b7e9aac36547cda8f974f6d2484c03</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,780,784,885,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23680982$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Skinner, Samuel O</creatorcontrib><creatorcontrib>Sepúlveda, Leonardo A</creatorcontrib><creatorcontrib>Xu, Heng</creatorcontrib><creatorcontrib>Golding, Ido</creatorcontrib><title>Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization</title><title>Nature protocols</title><addtitle>Nat Protoc</addtitle><addtitle>Nat Protoc</addtitle><description>We present a protocol for measuring the absolute number of mRNA molecules from a gene of interest in individual, chemically fixed
Escherichia coli
cells. A set of fluorescently labeled oligonucleotide probes is hybridized to the target mRNA, such that each mRNA molecule is decorated by a known number of fluorescent dyes. Cells are then imaged using fluorescence microscopy. The copy number of the target mRNA is estimated from the total intensity of fluorescent foci in the cell, rather than from counting discrete 'spots' as in other currently available protocols. Image analysis is performed using an automated algorithm. The measured mRNA copy number distribution obtained from many individual cells can be used to extract the parameters of stochastic gene activity, namely the frequency and size of transcription bursts from the gene of interest. The experimental procedure takes 2 d, with another 2–3 d typically required for image and data analysis.</description><subject>631/1647/2017/1947</subject><subject>631/1647/2217/457/649/2157</subject><subject>631/1647/245/2225</subject><subject>Algorithms</subject><subject>Analytical Chemistry</subject><subject>Automation</subject><subject>Biological Techniques</subject><subject>Cells</subject><subject>Computational Biology/Bioinformatics</subject><subject>Copy number</subject><subject>Data analysis</subject><subject>Dyes</subject><subject>E coli</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>Fluorescence</subject><subject>Fluorescence in situ hybridization</subject><subject>Fluorescence microscopy</subject><subject>Fluorescent dyes</subject><subject>Fluorescent indicators</subject><subject>Genetic aspects</subject><subject>Genetic variation</subject><subject>Hybridization</subject><subject>Image analysis</subject><subject>Image processing</subject><subject>In situ hybridization</subject><subject>In Situ Hybridization, Fluorescence - methods</subject><subject>Life Sciences</subject><subject>Measurement</subject><subject>Microarrays</subject><subject>Microscopy, Fluorescence</subject><subject>Oligonucleotide Probes - genetics</subject><subject>Oligonucleotides</subject><subject>Organic Chemistry</subject><subject>Physiological aspects</subject><subject>Probes</subject><subject>Protocol</subject><subject>RNA, Messenger - analysis</subject><subject>Stochasticity</subject><subject>Transcription</subject><issn>1754-2189</issn><issn>1750-2799</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNp9kl1vFCEUhidGY2v11ksziTcaM1sGGD5uTDabqk1qTWqNl4RhmF0aBlYYquuvl9nW2jWrgQCB57wcDm9RPK_BrAaIHbt18OMMghrNACEPisOaNqCClPOH2zWuYM34QfEkxisAMEWEPi4OICIMcAYPi-8ftYwpGLcsh4vzean8elO6NLQ6lMbl3plr0yVpy5OoVjoYtTIyU9aUSlsbyxSn2Gmwuhq81SpZXfY2-aCj0m6cZKIZU7natMF05qccjXdPi0e9tFE_u52Pii_vTi4XH6qzT-9PF_OzShHGxwpqRRrQkg7UqiWA1RA1rK0xoqihkiPS45ZqLqVCpMFUdZL1nOKedBAzrAA6Kt7e6K5TO-huSihIK9bBDDJshJdG7J44sxJLfy0wgLzhMAu8uhUI_lvScRSDidPTpdM-RVGjBgGMMSEZffkXeuVTcPl5AjYEI4gJ5f-jshakTf419odaSquFcb3P2anpajFHqCGQMjJpzfZQuXV6MMo73Zu8vxPweicgM6P-MS5lilGcfr7YZd_8m51ffl2c701FBR9j0P1djWsgJquKrVXFZFUBttV6cf9n7vDf3szA8Q0Q15M_dbhXqP2SvwCPJPNx</recordid><startdate>20130601</startdate><enddate>20130601</enddate><creator>Skinner, Samuel O</creator><creator>Sepúlveda, Leonardo A</creator><creator>Xu, Heng</creator><creator>Golding, Ido</creator><general>Nature Publishing Group UK</general><general>Nature Publishing Group</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>ATWCN</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7T5</scope><scope>7T7</scope><scope>7TM</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PATMY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20130601</creationdate><title>Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization</title><author>Skinner, Samuel O ; Sepúlveda, Leonardo A ; Xu, Heng ; Golding, Ido</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c689t-2ec650b6d01cb60812358b1437357a936f4b7e9aac36547cda8f974f6d2484c03</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>631/1647/2017/1947</topic><topic>631/1647/2217/457/649/2157</topic><topic>631/1647/245/2225</topic><topic>Algorithms</topic><topic>Analytical Chemistry</topic><topic>Automation</topic><topic>Biological Techniques</topic><topic>Cells</topic><topic>Computational Biology/Bioinformatics</topic><topic>Copy number</topic><topic>Data analysis</topic><topic>Dyes</topic><topic>E coli</topic><topic>Escherichia coli</topic><topic>Escherichia coli - genetics</topic><topic>Fluorescence</topic><topic>Fluorescence in situ hybridization</topic><topic>Fluorescence microscopy</topic><topic>Fluorescent dyes</topic><topic>Fluorescent indicators</topic><topic>Genetic aspects</topic><topic>Genetic variation</topic><topic>Hybridization</topic><topic>Image analysis</topic><topic>Image processing</topic><topic>In situ hybridization</topic><topic>In Situ Hybridization, Fluorescence - methods</topic><topic>Life Sciences</topic><topic>Measurement</topic><topic>Microarrays</topic><topic>Microscopy, Fluorescence</topic><topic>Oligonucleotide Probes - genetics</topic><topic>Oligonucleotides</topic><topic>Organic Chemistry</topic><topic>Physiological aspects</topic><topic>Probes</topic><topic>Protocol</topic><topic>RNA, Messenger - analysis</topic><topic>Stochasticity</topic><topic>Transcription</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Skinner, Samuel O</creatorcontrib><creatorcontrib>Sepúlveda, Leonardo A</creatorcontrib><creatorcontrib>Xu, Heng</creatorcontrib><creatorcontrib>Golding, Ido</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Middle School</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Immunology Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Nucleic Acids Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Environmental Science Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>Environmental Science Collection</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Nature protocols</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Skinner, Samuel O</au><au>Sepúlveda, Leonardo A</au><au>Xu, Heng</au><au>Golding, Ido</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization</atitle><jtitle>Nature protocols</jtitle><stitle>Nat Protoc</stitle><addtitle>Nat Protoc</addtitle><date>2013-06-01</date><risdate>2013</risdate><volume>8</volume><issue>6</issue><spage>1100</spage><epage>1113</epage><pages>1100-1113</pages><issn>1754-2189</issn><eissn>1750-2799</eissn><abstract>We present a protocol for measuring the absolute number of mRNA molecules from a gene of interest in individual, chemically fixed
Escherichia coli
cells. A set of fluorescently labeled oligonucleotide probes is hybridized to the target mRNA, such that each mRNA molecule is decorated by a known number of fluorescent dyes. Cells are then imaged using fluorescence microscopy. The copy number of the target mRNA is estimated from the total intensity of fluorescent foci in the cell, rather than from counting discrete 'spots' as in other currently available protocols. Image analysis is performed using an automated algorithm. The measured mRNA copy number distribution obtained from many individual cells can be used to extract the parameters of stochastic gene activity, namely the frequency and size of transcription bursts from the gene of interest. The experimental procedure takes 2 d, with another 2–3 d typically required for image and data analysis.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>23680982</pmid><doi>10.1038/nprot.2013.066</doi><tpages>14</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | 631/1647/2017/1947 631/1647/2217/457/649/2157 631/1647/245/2225 Algorithms Analytical Chemistry Automation Biological Techniques Cells Computational Biology/Bioinformatics Copy number Data analysis Dyes E coli Escherichia coli Escherichia coli - genetics Fluorescence Fluorescence in situ hybridization Fluorescence microscopy Fluorescent dyes Fluorescent indicators Genetic aspects Genetic variation Hybridization Image analysis Image processing In situ hybridization In Situ Hybridization, Fluorescence - methods Life Sciences Measurement Microarrays Microscopy, Fluorescence Oligonucleotide Probes - genetics Oligonucleotides Organic Chemistry Physiological aspects Probes Protocol RNA, Messenger - analysis Stochasticity Transcription |
| title | Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization |
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