Optimized procedure for high-throughput transcriptome profiling of small extracellular vesicles isolated from low volume serum samples
Small extracellular vesicles (EVs) contain various signaling molecules, thus playing a crucial role in cell-to-cell communication and emerging as a promising source of biomarkers. However, the lack of standardized procedures impedes their translation to clinical practice. Thus, we compared different...
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| Veröffentlicht in: | Clinical chemistry and laboratory medicine 2024-01, Vol.62 (1), p.157-167 |
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| creator | Vychytilova-Faltejskova, Petra Vilmanova, Sara Pifkova, Lucie Catela Ivković, Tina Mᶏdrzyk, Marie Jugas, Robin Machackova, Tana Kotoucek, Jan Sachlova, Milana Bohovicova, Lucia Stanek, Teodor Halamkova, Jana Kiss, Igor Slaby, Ondrej |
| description | Small extracellular vesicles (EVs) contain various signaling molecules, thus playing a crucial role in cell-to-cell communication and emerging as a promising source of biomarkers. However, the lack of standardized procedures impedes their translation to clinical practice. Thus, we compared different approaches for high-throughput analysis of small EVs transcriptome.
Small EVs were isolated from 150 μL of serum. Quality and quantity were assessed by dynamic light scattering, transmission electron microscopy, and Western blot. Comparison of RNA extraction efficiency was performed, and expression of selected genes was analyzed by RT-qPCR. Whole transcriptome analysis was done using microarrays.
Obtained data confirmed the suitability of size exclusion chromatography for isolation of small EVs. Analyses of gene expression showed the best results in case of samples isolated by Monarch Total RNA Miniprep Kit. Totally, 7,182 transcripts were identified to be deregulated between colorectal cancer patients and healthy controls. The majority of them were non-coding RNAs with more than 70 % being lncRNAs, while protein-coding genes represented the second most common gene biotype.
We have optimized the protocol for isolation of small EVs and their RNA from low volume of sera and confirmed the suitability of Clariom D Pico Assays for transcriptome profiling. |
| doi_str_mv | 10.1515/cclm-2023-0610 |
| format | Article |
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Small EVs were isolated from 150 μL of serum. Quality and quantity were assessed by dynamic light scattering, transmission electron microscopy, and Western blot. Comparison of RNA extraction efficiency was performed, and expression of selected genes was analyzed by RT-qPCR. Whole transcriptome analysis was done using microarrays.
Obtained data confirmed the suitability of size exclusion chromatography for isolation of small EVs. Analyses of gene expression showed the best results in case of samples isolated by Monarch Total RNA Miniprep Kit. Totally, 7,182 transcripts were identified to be deregulated between colorectal cancer patients and healthy controls. The majority of them were non-coding RNAs with more than 70 % being lncRNAs, while protein-coding genes represented the second most common gene biotype.
We have optimized the protocol for isolation of small EVs and their RNA from low volume of sera and confirmed the suitability of Clariom D Pico Assays for transcriptome profiling.</description><identifier>ISSN: 1434-6621</identifier><identifier>ISSN: 1437-4331</identifier><identifier>EISSN: 1437-4331</identifier><identifier>DOI: 10.1515/cclm-2023-0610</identifier><identifier>PMID: 37505924</identifier><language>eng</language><publisher>Germany: De Gruyter</publisher><subject>Biomarkers ; Cell interactions ; Chromatography, Gel ; Colorectal cancer ; Colorectal carcinoma ; Deregulation ; DNA microarrays ; Extracellular vesicles ; Extracellular Vesicles - genetics ; Extracellular Vesicles - metabolism ; Gene expression ; Gene Expression Profiling - methods ; Genes ; high-throughput expression profiling ; Humans ; Light scattering ; long non-coding RNAs ; Non-coding RNA ; Photon correlation spectroscopy ; Polymerase chain reaction ; Quality assessment ; Ribonucleic acid ; RNA ; Size exclusion chromatography ; small extracellular vesicles ; transcriptome ; Transcriptomes ; Transmission electron microscopy ; Vesicles</subject><ispartof>Clinical chemistry and laboratory medicine, 2024-01, Vol.62 (1), p.157-167</ispartof><rights>2023 the author(s), published by De Gruyter, Berlin/Boston.</rights><rights>2024. This work is published under http://creativecommons.org/licenses/by/4.0 (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c376t-b3fada421e6cd9975047298ce6bb96991fcbffce334d9a562814df0a813381a63</citedby><cites>FETCH-LOGICAL-c376t-b3fada421e6cd9975047298ce6bb96991fcbffce334d9a562814df0a813381a63</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.degruyter.com/document/doi/10.1515/cclm-2023-0610/pdf$$EPDF$$P50$$Gwalterdegruyter$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.degruyter.com/document/doi/10.1515/cclm-2023-0610/html$$EHTML$$P50$$Gwalterdegruyter$$Hfree_for_read</linktohtml><link.rule.ids>315,781,785,27929,27930,66759,68543</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/37505924$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Vychytilova-Faltejskova, Petra</creatorcontrib><creatorcontrib>Vilmanova, Sara</creatorcontrib><creatorcontrib>Pifkova, Lucie</creatorcontrib><creatorcontrib>Catela Ivković, Tina</creatorcontrib><creatorcontrib>Mᶏdrzyk, Marie</creatorcontrib><creatorcontrib>Jugas, Robin</creatorcontrib><creatorcontrib>Machackova, Tana</creatorcontrib><creatorcontrib>Kotoucek, Jan</creatorcontrib><creatorcontrib>Sachlova, Milana</creatorcontrib><creatorcontrib>Bohovicova, Lucia</creatorcontrib><creatorcontrib>Stanek, Teodor</creatorcontrib><creatorcontrib>Halamkova, Jana</creatorcontrib><creatorcontrib>Kiss, Igor</creatorcontrib><creatorcontrib>Slaby, Ondrej</creatorcontrib><title>Optimized procedure for high-throughput transcriptome profiling of small extracellular vesicles isolated from low volume serum samples</title><title>Clinical chemistry and laboratory medicine</title><addtitle>Clin Chem Lab Med</addtitle><description>Small extracellular vesicles (EVs) contain various signaling molecules, thus playing a crucial role in cell-to-cell communication and emerging as a promising source of biomarkers. However, the lack of standardized procedures impedes their translation to clinical practice. Thus, we compared different approaches for high-throughput analysis of small EVs transcriptome.
Small EVs were isolated from 150 μL of serum. Quality and quantity were assessed by dynamic light scattering, transmission electron microscopy, and Western blot. Comparison of RNA extraction efficiency was performed, and expression of selected genes was analyzed by RT-qPCR. Whole transcriptome analysis was done using microarrays.
Obtained data confirmed the suitability of size exclusion chromatography for isolation of small EVs. Analyses of gene expression showed the best results in case of samples isolated by Monarch Total RNA Miniprep Kit. Totally, 7,182 transcripts were identified to be deregulated between colorectal cancer patients and healthy controls. The majority of them were non-coding RNAs with more than 70 % being lncRNAs, while protein-coding genes represented the second most common gene biotype.
We have optimized the protocol for isolation of small EVs and their RNA from low volume of sera and confirmed the suitability of Clariom D Pico Assays for transcriptome profiling.</description><subject>Biomarkers</subject><subject>Cell interactions</subject><subject>Chromatography, Gel</subject><subject>Colorectal cancer</subject><subject>Colorectal carcinoma</subject><subject>Deregulation</subject><subject>DNA microarrays</subject><subject>Extracellular vesicles</subject><subject>Extracellular Vesicles - genetics</subject><subject>Extracellular Vesicles - metabolism</subject><subject>Gene expression</subject><subject>Gene Expression Profiling - methods</subject><subject>Genes</subject><subject>high-throughput expression profiling</subject><subject>Humans</subject><subject>Light scattering</subject><subject>long non-coding RNAs</subject><subject>Non-coding RNA</subject><subject>Photon correlation spectroscopy</subject><subject>Polymerase chain reaction</subject><subject>Quality assessment</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>Size exclusion chromatography</subject><subject>small extracellular vesicles</subject><subject>transcriptome</subject><subject>Transcriptomes</subject><subject>Transmission electron microscopy</subject><subject>Vesicles</subject><issn>1434-6621</issn><issn>1437-4331</issn><issn>1437-4331</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2024</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkTtvFTEQRi0EIiHQUiJLNDQb7PVj1xINinhJkdJAvfJ6x_c6sq8XPxLCD-B34-UGkBCVpzjzeWYOQs8pOaeCitfG-ND1pGcdkZQ8QKeUs6HjjNGHv2reSdnTE_Qk52tCqBB8eIxO2CCIUD0_RT-u1uKC-w4LXlM0sNQE2MaE926378o-xbrbr7XgkvQhm-TWEgNsrHXeHXY4WpyD9h7Dt4YY8L56nfANZGc8ZOxy9Lq0eJtiwD7e4pvoa4vIkGrAWYe1YU_RI6t9hmf37xn68v7d54uP3eXVh08Xby87wwZZuplZvWjeU5BmUaptwYdejQbkPCupFLVmttYAY3xRWsh-pHyxRI-UsZFqyc7Qq2NuW-BrhVym4PI2tD5ArHnqR8Hb0QghDX35D3odazq06Rql2NBursZGnR8pk2LOCey0Jhd0upsomTZD02Zo2gxNm6HW8OI-ts4Blj_4byUNeHMEbrUvkBbYpXrXir_f_z-5aaZiYD8BoP6juQ</recordid><startdate>20240126</startdate><enddate>20240126</enddate><creator>Vychytilova-Faltejskova, Petra</creator><creator>Vilmanova, Sara</creator><creator>Pifkova, Lucie</creator><creator>Catela Ivković, Tina</creator><creator>Mᶏdrzyk, Marie</creator><creator>Jugas, Robin</creator><creator>Machackova, Tana</creator><creator>Kotoucek, Jan</creator><creator>Sachlova, Milana</creator><creator>Bohovicova, Lucia</creator><creator>Stanek, Teodor</creator><creator>Halamkova, Jana</creator><creator>Kiss, Igor</creator><creator>Slaby, Ondrej</creator><general>De Gruyter</general><general>Walter De Gruyter & Company</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>7QO</scope><scope>7T7</scope><scope>7TK</scope><scope>7U7</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20240126</creationdate><title>Optimized procedure for high-throughput transcriptome profiling of small extracellular vesicles isolated from low volume serum samples</title><author>Vychytilova-Faltejskova, Petra ; 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However, the lack of standardized procedures impedes their translation to clinical practice. Thus, we compared different approaches for high-throughput analysis of small EVs transcriptome.
Small EVs were isolated from 150 μL of serum. Quality and quantity were assessed by dynamic light scattering, transmission electron microscopy, and Western blot. Comparison of RNA extraction efficiency was performed, and expression of selected genes was analyzed by RT-qPCR. Whole transcriptome analysis was done using microarrays.
Obtained data confirmed the suitability of size exclusion chromatography for isolation of small EVs. Analyses of gene expression showed the best results in case of samples isolated by Monarch Total RNA Miniprep Kit. Totally, 7,182 transcripts were identified to be deregulated between colorectal cancer patients and healthy controls. The majority of them were non-coding RNAs with more than 70 % being lncRNAs, while protein-coding genes represented the second most common gene biotype.
We have optimized the protocol for isolation of small EVs and their RNA from low volume of sera and confirmed the suitability of Clariom D Pico Assays for transcriptome profiling.</abstract><cop>Germany</cop><pub>De Gruyter</pub><pmid>37505924</pmid><doi>10.1515/cclm-2023-0610</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Biomarkers Cell interactions Chromatography, Gel Colorectal cancer Colorectal carcinoma Deregulation DNA microarrays Extracellular vesicles Extracellular Vesicles - genetics Extracellular Vesicles - metabolism Gene expression Gene Expression Profiling - methods Genes high-throughput expression profiling Humans Light scattering long non-coding RNAs Non-coding RNA Photon correlation spectroscopy Polymerase chain reaction Quality assessment Ribonucleic acid RNA Size exclusion chromatography small extracellular vesicles transcriptome Transcriptomes Transmission electron microscopy Vesicles |
| title | Optimized procedure for high-throughput transcriptome profiling of small extracellular vesicles isolated from low volume serum samples |
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