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In Situ Hybridization and Cytometry for Single Cell Measurements
In Situ Hybridization and Cytometry for Single Cell Measurements
상세정보
- 자료유형
- 학위논문 서양
- 최종처리일시
- 20260202103041
- ISBN
- 9798280751422
- DDC
- 610
- 저자명
- Foyt, Daniel.
- 서명/저자
- In Situ Hybridization and Cytometry for Single Cell Measurements
- 발행사항
- [Sl] : University of California, San Francisco, 2025
- 발행사항
- Ann Arbor : ProQuest Dissertations & Theses, 2025
- 형태사항
- 109 p
- 주기사항
- Source: Dissertations Abstracts International, Volume: 86-12, Section: B.
- 주기사항
- Includes supplementary digital materials.
- 주기사항
- Advisor: Huang, Bo.
- 학위논문주기
- Thesis (Ph.D.)--University of California, San Francisco, 2025.
- 초록/해제
- 요약Advancements in single-cell biology have revolutionized our understanding of complex biological systems by enabling high-throughput measurements of DNA, RNA, and proteins. This dissertation presents three innovative methodologies that advance single-cell analysis across these biomolecular domains.DNA: The FMR1 gene, essential for synaptic plasticity, contains a CGG repeat region where expansions (200 repeats) cause Fragile X Syndrome. Existing methods to measure expansion lack single-cell resolution and spatial context. To address this, I developed CGG FISH, an RNA-FISH-based approach that quantifies CGG repeats by fluorescence intensity. Tested in humanized mouse models and human fibroblast cell lines, CGG FISH distinguished repeat lengths but faced challenges at low expression levels. Simulations revealed variability in probe binding and repeat heterogeneity. While offering insights into cell-to-cell variability, CGG FISH requires complementary methods to achieve finer resolution for small repeat differences.RNA: Single-cell RNA sequencing suffers from inefficiencies in reverse transcription, especially for low-abundance RNAs. Inspired by single molecule FISH, I developed HybriSeq, a method combining in situ hybridization, ligation, and combinatorial indexing to enable highly sensitive, targeted, and scalable RNA profiling. HybriSeq achieves high specificity and sensitivity by amplifying signals linearly with multiple probes to reduce noise. This technique offers a robust solution for high-throughput single-cell RNA analysis with improved scalability.Proteins: To enhance microscopy-based cytometry, I developed a method using partial trypsinization and the Cellpose algorithm for precise segmentation. This approach improves accuracy across cell densities, maintains spatial context, and excels in signal fidelity compared to flow cytometry. Its utility was demonstrated in optimizing prime editing for gene tagging, showcasing its potential in genetic engineering studies.Together, these methods address key challenges in single-cell analysis, expanding our ability to study cellular heterogeneity and biological complexity.
- 일반주제명
- Bioengineering
- 일반주제명
- Cellular biology
- 일반주제명
- Genetics
- 키워드
- Cytometry
- 키워드
- HybriSeq
- 키워드
- Single cell
- 키워드
- RNA analysis
- 기타저자
- University of California, San Francisco Bioengineering
- 기본자료저록
- Dissertations Abstracts International. 86-12B.
- 전자적 위치 및 접속
- 로그인 후 원문을 볼 수 있습니다.
MARC
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■00520260202103041
■006m o d
■007cr#unu||||||||
■020 ▼a9798280751422
■035 ▼a(MiAaPQ)AAI31847696
■040 ▼aMiAaPQ▼cMiAaPQ
■0820 ▼a610
■1001 ▼aFoyt, Daniel.▼0(orcid)0000-0002-9897-4692
■24510▼aIn Situ Hybridization and Cytometry for Single Cell Measurements
■260 ▼a[Sl]▼bUniversity of California, San Francisco▼c2025
■260 1▼aAnn Arbor▼bProQuest Dissertations & Theses▼c2025
■300 ▼a109 p
■500 ▼aSource: Dissertations Abstracts International, Volume: 86-12, Section: B.
■500 ▼aIncludes supplementary digital materials.
■500 ▼aAdvisor: Huang, Bo.
■5021 ▼aThesis (Ph.D.)--University of California, San Francisco, 2025.
■520 ▼aAdvancements in single-cell biology have revolutionized our understanding of complex biological systems by enabling high-throughput measurements of DNA, RNA, and proteins. This dissertation presents three innovative methodologies that advance single-cell analysis across these biomolecular domains.DNA: The FMR1 gene, essential for synaptic plasticity, contains a CGG repeat region where expansions (200 repeats) cause Fragile X Syndrome. Existing methods to measure expansion lack single-cell resolution and spatial context. To address this, I developed CGG FISH, an RNA-FISH-based approach that quantifies CGG repeats by fluorescence intensity. Tested in humanized mouse models and human fibroblast cell lines, CGG FISH distinguished repeat lengths but faced challenges at low expression levels. Simulations revealed variability in probe binding and repeat heterogeneity. While offering insights into cell-to-cell variability, CGG FISH requires complementary methods to achieve finer resolution for small repeat differences.RNA: Single-cell RNA sequencing suffers from inefficiencies in reverse transcription, especially for low-abundance RNAs. Inspired by single molecule FISH, I developed HybriSeq, a method combining in situ hybridization, ligation, and combinatorial indexing to enable highly sensitive, targeted, and scalable RNA profiling. HybriSeq achieves high specificity and sensitivity by amplifying signals linearly with multiple probes to reduce noise. This technique offers a robust solution for high-throughput single-cell RNA analysis with improved scalability.Proteins: To enhance microscopy-based cytometry, I developed a method using partial trypsinization and the Cellpose algorithm for precise segmentation. This approach improves accuracy across cell densities, maintains spatial context, and excels in signal fidelity compared to flow cytometry. Its utility was demonstrated in optimizing prime editing for gene tagging, showcasing its potential in genetic engineering studies.Together, these methods address key challenges in single-cell analysis, expanding our ability to study cellular heterogeneity and biological complexity.
■590 ▼aSchool code: 0034.
■650 4▼aBioengineering
■650 4▼aCellular biology
■650 4▼aGenetics
■653 ▼aCytometry
■653 ▼aHybriSeq
■653 ▼aSingle cell
■653 ▼aRNA analysis
■690 ▼a0202
■690 ▼a0379
■690 ▼a0369
■71020▼aUniversity of California, San Francisco▼bBioengineering.
■7730 ▼tDissertations Abstracts International▼g86-12B.
■790 ▼a0034
■791 ▼aPh.D.
■792 ▼a2025
■793 ▼aEnglish
■85640▼uhttp://www.riss.kr/pdu/ddodLink.do?id=T17356813▼nKERIS▼z이 자료의 원문은 한국교육학술정보원에서 제공합니다.
