Microfluidic fractionation and analysis of cytoplasmic versus nuclear acids in single cells
Abstract/Contents
- Abstract
- Single-cell gene expression studies have matured and are now widely used to uncover cell-to-cell variability, transcript processing and inhomogeneous response to external stimuli. Despite great progress, significant challenges remain including understanding fundamental questions of human genome transcription, gene expression ranges, localization, and processing destinies of RNAs. Eukaryotic cells make different types of primary and processed transcripts, which are either exclusively found in particular sub-cellular compartments or dispersed throughout the whole cell. These sub-cellular localizations of RNAs are weakly understood at a single-cell level, and are important for fully realizing gene functions. Understanding sub-cellular transcript localization is particularly important for studying the process of splicing, a pathway during which introns of pre-messenger RNA's are excised and exons are stitched together to form mature mRNA transcript. We here introduce a single-cell-isotachophoresis (sc-ITP) method to analyze co-transcriptional and alternative splicing in reference lymphoblastoid cell line (LCL-Snyder), and two sub-lines of CML cells (K562-ATCC and K562-ENCODE). The method is unique in that we physically separate the contents of cell nucleus from those of the cytoplasm and analyze independently. It allows for rapid, electric-field-based selective lysis of cytoplasmic membrane (leaving nucleus intact); and then purification and simultaneous fractionation of total RNA in cytosol (cyt-RNA) and total RNA in the nucleus (nuc-RNA) from single cells with no intra-compartment cross-contamination. We then use our system and state-of-the-art NGS technologies to perform single-cell nuclear and cytoplasmic RNA-seq to study fundamental questions of human genome transcription, differential gene expression, localization, and processing destinies of RNAs for various cell types, disease, and differentiation states. We evaluate the distribution of whole transcriptome gene features and gene expression of precursor and processed transcripts and find that there are considerable differences in expression levels across sub-cellular compartments and among individual cells. We then evaluate cell-to-cell variability of the highly expressed GAPDH housekeeping gene in alternative splicing between the lymphoblastoid and leukaemia cells using sequencing-specific probes and RT-qPCR. The data suggest evidence of significant distinction in splicing patterns. By analyzing nuclear and cytoplasmic compartments of a single cell individually at a whole transcriptome level, we achieve an unprecedented precision in splicing quantification. Together, our results describe an experimental method for single-cell fractionation and an analytical tool for gene and isoform expression analysis in rare cell types, cell differentiation and disease states.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2015 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Milanova, Denitsa |
|---|---|
| Associated with | Stanford University, Department of Mechanical Engineering. |
| Primary advisor | Santiago, Juan G |
| Thesis advisor | Santiago, Juan G |
| Thesis advisor | Quake, Stephen Ronald |
| Thesis advisor | Snyder, Michael, Ph. D |
| Advisor | Quake, Stephen Ronald |
| Advisor | Snyder, Michael, Ph. D |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Denitsa Milanova. |
|---|---|
| Note | Submitted to the Department of Mechanical Engineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2015. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2015 by Denitsa Milanova Milanova
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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