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Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization
Published on: May 15, 2018
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Low-Volume On-Chip Single-Cell Whole Genome Amplification for Multiple Subsequent Analyses
Thomas Kroneis1,2, Shukun Chen3, Amin El-Heliebi3
1Research Unit for Single Cell Analysis, Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Harrachgasse 21, Graz, 8010, Austria. thomas.kroneis@medunigraz.at.
Methods in Molecular Biology (Clifton, N.J.)
|September 17, 2015
Summary
This study presents a new protocol for isolating and amplifying DNA from single cells using laser microdissection and isothermal whole genome amplification. This method enables multiple downstream analyses from rare cells, improving genetic research.
Area of Science:
- Genomics
- Molecular Biology
- Cell Biology
Background:
- Single-cell DNA analysis is crucial but challenging due to the large human genome requiring pre-amplification.
- Existing methods struggle with rare cells mixed with abundant background cells.
- Isothermal whole genome amplification (WGA) is effective for amplifying long DNA from single cells.
Purpose of the Study:
- To develop a protocol for isolating and amplifying DNA from single, rare cells for downstream genomic analyses.
- To enable multiple analyses from limited single-cell samples.
- To overcome challenges associated with analyzing rare cells in complex mixtures.
Main Methods:
- Candidate target cells are identified using specific staining.
- Cells are isolated using laser microdissection and pressure catapulting (LMPC).
- Isolated single cells undergo isothermal whole genome amplification on a microliter reaction slide.
Main Results:
- The protocol successfully isolates and amplifies DNA from single cells.
- The process is monitored on a microliter slide, allowing real-time observation.
- Amplification occurs in under 2 μL, preserving DNA for multiple analyses.
Conclusions:
- This protocol provides a robust method for single-cell DNA isolation and amplification.
- It is particularly valuable for analyzing rare cells in complex biological samples.
- The amplified DNA is suitable for various downstream genomic applications, advancing single-cell research.
