Effective microtissue RNA extraction coupled with Smart-seq2 for reproducible and robust spatial transcriptome analysis
Spatial transcriptomics is useful for understanding the molecular organization of a tissue and providing insights into cellular function in a morphological context. In order to obtain reproducible results in spatial transcriptomics, we have to maintain tissue morphology and RNA molecule stability du...
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| Veröffentlicht in: | Scientific reports 2020-04, Vol.10 (1), p.7083-7083, Article 7083 |
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| creator | Yamazaki, Miki Hosokawa, Masahito Arikawa, Koji Takahashi, Kiyofumi Sakanashi, Chikako Yoda, Takuya Matsunaga, Hiroko Takeyama, Haruko |
| description | Spatial transcriptomics is useful for understanding the molecular organization of a tissue and providing insights into cellular function in a morphological context. In order to obtain reproducible results in spatial transcriptomics, we have to maintain tissue morphology and RNA molecule stability during the image acquisition and biomolecule collection processes. Here, we developed a tissue processing method for robust and reproducible RNA-seq from tissue microdissection samples. In this method, we suppressed RNA degradation in fresh-frozen tissue specimens by dehydration fixation and effectively collected a small amount of RNA molecules from microdissection samples by magnetic beads. We demonstrated the spatial transcriptome analysis of the mouse liver and brain in serial microdissection samples (100 μm in a diameter and 10 μm in thickness) produced by a microdissection punching system. Using our method, we could prevent RNA degradation at room temperature and effectively produce a sequencing library with Smart-seq2. This resulted in reproducible sequence read mapping in exon regions and the detection of more than 2000 genes compared to non-fixed samples in the RNA-seq analysis. Our method would be applied to various transcriptome analyses, providing the information for region specific gene expression in tissue specimens. |
| doi_str_mv | 10.1038/s41598-020-63495-6 |
| format | Article |
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In order to obtain reproducible results in spatial transcriptomics, we have to maintain tissue morphology and RNA molecule stability during the image acquisition and biomolecule collection processes. Here, we developed a tissue processing method for robust and reproducible RNA-seq from tissue microdissection samples. In this method, we suppressed RNA degradation in fresh-frozen tissue specimens by dehydration fixation and effectively collected a small amount of RNA molecules from microdissection samples by magnetic beads. We demonstrated the spatial transcriptome analysis of the mouse liver and brain in serial microdissection samples (100 μm in a diameter and 10 μm in thickness) produced by a microdissection punching system. Using our method, we could prevent RNA degradation at room temperature and effectively produce a sequencing library with Smart-seq2. This resulted in reproducible sequence read mapping in exon regions and the detection of more than 2000 genes compared to non-fixed samples in the RNA-seq analysis. Our method would be applied to various transcriptome analyses, providing the information for region specific gene expression in tissue specimens.</description><identifier>ISSN: 2045-2322</identifier><identifier>EISSN: 2045-2322</identifier><identifier>DOI: 10.1038/s41598-020-63495-6</identifier><identifier>PMID: 32341421</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>631/1647/2217/2018 ; 631/61/212/2019 ; 631/61/514/1949 ; Animals ; Dehydration ; Gene expression ; Gene Expression Profiling ; Gene mapping ; Humanities and Social Sciences ; Male ; Mice ; Mice, Inbred ICR ; Microdissection ; multidisciplinary ; Reproducibility of Results ; Ribonucleic acid ; RNA ; RNA-Seq ; Science ; Science (multidisciplinary) ; Spatial analysis ; Tissues ; Transcriptomics</subject><ispartof>Scientific reports, 2020-04, Vol.10 (1), p.7083-7083, Article 7083</ispartof><rights>The Author(s) 2020</rights><rights>The Author(s) 2020. 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In order to obtain reproducible results in spatial transcriptomics, we have to maintain tissue morphology and RNA molecule stability during the image acquisition and biomolecule collection processes. Here, we developed a tissue processing method for robust and reproducible RNA-seq from tissue microdissection samples. In this method, we suppressed RNA degradation in fresh-frozen tissue specimens by dehydration fixation and effectively collected a small amount of RNA molecules from microdissection samples by magnetic beads. We demonstrated the spatial transcriptome analysis of the mouse liver and brain in serial microdissection samples (100 μm in a diameter and 10 μm in thickness) produced by a microdissection punching system. Using our method, we could prevent RNA degradation at room temperature and effectively produce a sequencing library with Smart-seq2. 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In order to obtain reproducible results in spatial transcriptomics, we have to maintain tissue morphology and RNA molecule stability during the image acquisition and biomolecule collection processes. Here, we developed a tissue processing method for robust and reproducible RNA-seq from tissue microdissection samples. In this method, we suppressed RNA degradation in fresh-frozen tissue specimens by dehydration fixation and effectively collected a small amount of RNA molecules from microdissection samples by magnetic beads. We demonstrated the spatial transcriptome analysis of the mouse liver and brain in serial microdissection samples (100 μm in a diameter and 10 μm in thickness) produced by a microdissection punching system. Using our method, we could prevent RNA degradation at room temperature and effectively produce a sequencing library with Smart-seq2. 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| subjects | 631/1647/2217/2018 631/61/212/2019 631/61/514/1949 Animals Dehydration Gene expression Gene Expression Profiling Gene mapping Humanities and Social Sciences Male Mice Mice, Inbred ICR Microdissection multidisciplinary Reproducibility of Results Ribonucleic acid RNA RNA-Seq Science Science (multidisciplinary) Spatial analysis Tissues Transcriptomics |
| title | Effective microtissue RNA extraction coupled with Smart-seq2 for reproducible and robust spatial transcriptome analysis |
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