2022 Fiscal Year Final Research Report
Development of histology-associated high-quality single-cell RNA-sequencing method in ovarian folliculogenesis
| Project Area | Ensuring integrity in gametogenesis |
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| Project/Area Number |
18H05553
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| Research Category |
Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
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| Allocation Type | Single-year Grants |
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| Review Section |
Biological Sciences
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| Research Institution | Nara Medical University |
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Principal Investigator |
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| Project Period (FY) |
2018-06-29 – 2023-03-31
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| Keywords | シングルセルトランスクリプトーム / 組織切片 / RNA-seq / 空間トランスクリプトーム / 生殖細胞 / 配偶子形成 |
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| Outline of Final Research Achievements |
We developed DRaqL (direct RNA recovery and quenching for LCM), an experimental approach for efficient lysis of single cells isolated by LCM from alcohol-and formalin-fixed sections without RNA purification. Single-cell RNA-seq combined with DRaqL allowed transcriptomic profiling from alcohol-fixed sections with efficiency comparable to freshly dissociated cells, together with exon-exon junction profiling. Combination of DRaqL and protease treatment enabled robust analysis from strongly fixed-formalin tissue sections. Applying this method to mouse ovarian sections, we revealed a transcriptomic continuum of growing oocytes quantitatively associated with oocyte size, and detected oocyte-specific splice isoforms. We also identified genes that were differentially expressed in granulosa cells in association with the histological affiliations of granulosa cells to the oocytes, suggesting distinct epigenetic regulations and cell-cycle activities governing the germ-soma relationship.
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| Free Research Field |
発生生物学
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| Academic Significance and Societal Importance of the Research Achievements |
組織切片から採取した単一の細胞の遺伝子発現を精密におこなうことができる技術を開発した。これにより高精度な組織学的な解析で細胞の形態的・位置的情報を得た後に、正確な遺伝発現情報を得ることができるようになった。広く行われる組織切片全体にわたる空間トランスクリプトーム解析技術と比較して、多数の切片に渡る形態的に同定可能な細胞に対して高感度な解析が可能であるという相補的な特性を持つ。この手法を用いてマウス卵巣中の卵母細胞のサイズと遺伝子発現の間の関係の多様性を見出した。これは卵母細胞の品質管理機構に関する新しいアプローチを提供すると期待される。
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