Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ
RNA sequencing (RNA-seq) is a sensitive and accurate method for quantifying gene expression. Small samples or those whose RNA is degraded, such as formalin-fixed paraffin-embedded (FFPE) tissue, remain challenging to study with nonspecialized RNA-seq protocols. Here, we present a new method, Smart-3...
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| Veröffentlicht in: | Genome research 2019-11, Vol.29 (11), p.1816-1825 |
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| container_title | Genome research |
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| creator | Foley, Joseph W Zhu, Chunfang Jolivet, Philippe Zhu, Shirley X Lu, Peipei Meaney, Michael J West, Robert B |
| description | RNA sequencing (RNA-seq) is a sensitive and accurate method for quantifying gene expression. Small samples or those whose RNA is degraded, such as formalin-fixed paraffin-embedded (FFPE) tissue, remain challenging to study with nonspecialized RNA-seq protocols. Here, we present a new method, Smart-3SEQ, that accurately quantifies transcript abundance even with small amounts of total RNA and effectively characterizes small samples extracted by laser-capture microdissection (LCM) from FFPE tissue. We also obtain distinct biological profiles from FFPE single cells, which have been impossible to study with previous RNA-seq protocols, and we use these data to identify possible new macrophage phenotypes associated with the tumor microenvironment. We propose Smart-3SEQ as a highly cost-effective method to enable large gene expression profiling experiments unconstrained by sample size and tissue availability. In particular, Smart-3SEQ's compatibility with FFPE tissue unlocks an enormous number of archived clinical samples; combined with LCM it allows unprecedented studies of small cell populations and single cells isolated by their in situ context. |
| doi_str_mv | 10.1101/gr.234807.118 |
| format | Article |
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Small samples or those whose RNA is degraded, such as formalin-fixed paraffin-embedded (FFPE) tissue, remain challenging to study with nonspecialized RNA-seq protocols. Here, we present a new method, Smart-3SEQ, that accurately quantifies transcript abundance even with small amounts of total RNA and effectively characterizes small samples extracted by laser-capture microdissection (LCM) from FFPE tissue. We also obtain distinct biological profiles from FFPE single cells, which have been impossible to study with previous RNA-seq protocols, and we use these data to identify possible new macrophage phenotypes associated with the tumor microenvironment. We propose Smart-3SEQ as a highly cost-effective method to enable large gene expression profiling experiments unconstrained by sample size and tissue availability. In particular, Smart-3SEQ's compatibility with FFPE tissue unlocks an enormous number of archived clinical samples; combined with LCM it allows unprecedented studies of small cell populations and single cells isolated by their in situ context.</description><identifier>ISSN: 1088-9051</identifier><identifier>EISSN: 1549-5469</identifier><identifier>DOI: 10.1101/gr.234807.118</identifier><identifier>PMID: 31519740</identifier><language>eng</language><publisher>United States: Cold Spring Harbor Laboratory Press</publisher><subject>Gene expression ; Gene Expression Profiling - methods ; Humans ; Laser Capture Microdissection - methods ; Macrophages ; Macrophages - metabolism ; Method ; Paraffin ; Phenotypes ; Population studies ; Reproducibility of Results ; Ribonucleic acid ; RNA ; Sequence Analysis, RNA - methods ; Single-Cell Analysis - methods ; Transcription ; Tumor Microenvironment</subject><ispartof>Genome research, 2019-11, Vol.29 (11), p.1816-1825</ispartof><rights>2019 Foley et al.; Published by Cold Spring Harbor Laboratory Press.</rights><rights>Copyright Cold Spring Harbor Laboratory Press Nov 2019</rights><rights>2019</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c415t-deaa26ea921d236322f95e540307ba10b7ad1e5f507f0d372f8bb4d7c6ea05ef3</citedby><cites>FETCH-LOGICAL-c415t-deaa26ea921d236322f95e540307ba10b7ad1e5f507f0d372f8bb4d7c6ea05ef3</cites><orcidid>0000-0002-1579-5286</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836736/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836736/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,881,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31519740$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Foley, Joseph W</creatorcontrib><creatorcontrib>Zhu, Chunfang</creatorcontrib><creatorcontrib>Jolivet, Philippe</creatorcontrib><creatorcontrib>Zhu, Shirley X</creatorcontrib><creatorcontrib>Lu, Peipei</creatorcontrib><creatorcontrib>Meaney, Michael J</creatorcontrib><creatorcontrib>West, Robert B</creatorcontrib><title>Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ</title><title>Genome research</title><addtitle>Genome Res</addtitle><description>RNA sequencing (RNA-seq) is a sensitive and accurate method for quantifying gene expression. Small samples or those whose RNA is degraded, such as formalin-fixed paraffin-embedded (FFPE) tissue, remain challenging to study with nonspecialized RNA-seq protocols. Here, we present a new method, Smart-3SEQ, that accurately quantifies transcript abundance even with small amounts of total RNA and effectively characterizes small samples extracted by laser-capture microdissection (LCM) from FFPE tissue. We also obtain distinct biological profiles from FFPE single cells, which have been impossible to study with previous RNA-seq protocols, and we use these data to identify possible new macrophage phenotypes associated with the tumor microenvironment. We propose Smart-3SEQ as a highly cost-effective method to enable large gene expression profiling experiments unconstrained by sample size and tissue availability. In particular, Smart-3SEQ's compatibility with FFPE tissue unlocks an enormous number of archived clinical samples; combined with LCM it allows unprecedented studies of small cell populations and single cells isolated by their in situ context.</description><subject>Gene expression</subject><subject>Gene Expression Profiling - methods</subject><subject>Humans</subject><subject>Laser Capture Microdissection - methods</subject><subject>Macrophages</subject><subject>Macrophages - metabolism</subject><subject>Method</subject><subject>Paraffin</subject><subject>Phenotypes</subject><subject>Population studies</subject><subject>Reproducibility of Results</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>Sequence Analysis, RNA - methods</subject><subject>Single-Cell Analysis - methods</subject><subject>Transcription</subject><subject>Tumor Microenvironment</subject><issn>1088-9051</issn><issn>1549-5469</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpdkclLxTAQxoMoLk-PXiXgxUt1kjRdLoKIGwgi6jmk7eS9SNvUpHX5783jqainyTC_zPJ9hOwzOGYM2MncH3ORFpDHtFgj20ymZSLTrFyPbyiKpATJtshOCM8AEMlik2wJJlmZp7BNhivskeL74DEE63o6eGdsa_s5dYaGGFukNbZtoMa7jmpfL-yrbuloQ5iQvtlxQVsd0Ce1HsbJI-1s7V0Ty1iPy466b-hDp_2YiIeL-12yYXQbcO8rzsjT5cXj-XVye3d1c352m9Qpk2PSoNY8Q11y1nCRCc5NKVGmICCvNIMq1w1DaSTkBhqRc1NUVdrkdfwDEo2YkdNV32GqOmxq7EevWzV4G1f5UE5b9bfS24Wau1eVFSLL48QZOfpq4N3LhGFUnQ1LJXSPbgqK8xJK4CzCM3L4D312k-_jeYpHqUHKyEUqWVFRnhA8mp9lGKill2ru1crLmBaRP_h9wQ_9bZ74BBXDnGg</recordid><startdate>20191101</startdate><enddate>20191101</enddate><creator>Foley, Joseph W</creator><creator>Zhu, Chunfang</creator><creator>Jolivet, Philippe</creator><creator>Zhu, Shirley X</creator><creator>Lu, Peipei</creator><creator>Meaney, Michael J</creator><creator>West, Robert B</creator><general>Cold Spring Harbor Laboratory Press</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>7TM</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-1579-5286</orcidid></search><sort><creationdate>20191101</creationdate><title>Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ</title><author>Foley, Joseph W ; Zhu, Chunfang ; Jolivet, Philippe ; Zhu, Shirley X ; Lu, Peipei ; Meaney, Michael J ; West, Robert B</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c415t-deaa26ea921d236322f95e540307ba10b7ad1e5f507f0d372f8bb4d7c6ea05ef3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>Gene expression</topic><topic>Gene Expression Profiling - methods</topic><topic>Humans</topic><topic>Laser Capture Microdissection - methods</topic><topic>Macrophages</topic><topic>Macrophages - metabolism</topic><topic>Method</topic><topic>Paraffin</topic><topic>Phenotypes</topic><topic>Population studies</topic><topic>Reproducibility of Results</topic><topic>Ribonucleic acid</topic><topic>RNA</topic><topic>Sequence Analysis, RNA - methods</topic><topic>Single-Cell Analysis - methods</topic><topic>Transcription</topic><topic>Tumor Microenvironment</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Foley, Joseph W</creatorcontrib><creatorcontrib>Zhu, Chunfang</creatorcontrib><creatorcontrib>Jolivet, Philippe</creatorcontrib><creatorcontrib>Zhu, Shirley X</creatorcontrib><creatorcontrib>Lu, Peipei</creatorcontrib><creatorcontrib>Meaney, Michael J</creatorcontrib><creatorcontrib>West, Robert B</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Genome research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Foley, Joseph W</au><au>Zhu, Chunfang</au><au>Jolivet, Philippe</au><au>Zhu, Shirley X</au><au>Lu, Peipei</au><au>Meaney, Michael J</au><au>West, Robert B</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ</atitle><jtitle>Genome research</jtitle><addtitle>Genome Res</addtitle><date>2019-11-01</date><risdate>2019</risdate><volume>29</volume><issue>11</issue><spage>1816</spage><epage>1825</epage><pages>1816-1825</pages><issn>1088-9051</issn><eissn>1549-5469</eissn><abstract>RNA sequencing (RNA-seq) is a sensitive and accurate method for quantifying gene expression. Small samples or those whose RNA is degraded, such as formalin-fixed paraffin-embedded (FFPE) tissue, remain challenging to study with nonspecialized RNA-seq protocols. Here, we present a new method, Smart-3SEQ, that accurately quantifies transcript abundance even with small amounts of total RNA and effectively characterizes small samples extracted by laser-capture microdissection (LCM) from FFPE tissue. We also obtain distinct biological profiles from FFPE single cells, which have been impossible to study with previous RNA-seq protocols, and we use these data to identify possible new macrophage phenotypes associated with the tumor microenvironment. We propose Smart-3SEQ as a highly cost-effective method to enable large gene expression profiling experiments unconstrained by sample size and tissue availability. 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| subjects | Gene expression Gene Expression Profiling - methods Humans Laser Capture Microdissection - methods Macrophages Macrophages - metabolism Method Paraffin Phenotypes Population studies Reproducibility of Results Ribonucleic acid RNA Sequence Analysis, RNA - methods Single-Cell Analysis - methods Transcription Tumor Microenvironment |
| title | Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ |
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