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Amplification of multiple genomic loci from single cells isolated by laser micro-dissection of tissues.

Frumkin D, Wasserstrom A, Itzkovitz S, Harmelin A, Rechavi G, Shapiro E - BMC Biotechnol. (2008)

Bottom Line: In cells obtained from fresh tissue sections, 64.5% of alleles successfully amplified to approximately 700000 copies each, and mitochondrial DNA was amplified successfully in all cells.Multiplex PCR amplification and analysis of cells from pre-stored sections yielded significantly poorer results.Comprehensive genomic analysis of single cells from stained tissue sections opens new research opportunities for cell lineage and depth analyses, genome-wide mutation surveys, and other single cell assays.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel. dan.frumkin@weizmann.ac.il

ABSTRACT

Background: Whole genome amplification (WGA) and laser assisted micro-dissection represent two recently developed technologies that can greatly advance biological and medical research. WGA allows the analysis of multiple genomic loci from a single genome and has been performed on single cells from cell suspensions and from enzymatically-digested tissues. Laser micro-dissection makes it possible to isolate specific single cells from heterogeneous tissues.

Results: Here we applied for the first time WGA on laser micro-dissected single cells from stained tissue sections, and developed a protocol for sequentially performing the two procedures. The combined procedure allows correlating the cell's genome with its natural morphology and precise anatomical position. From each cell we amplified 122 genomic and mitochondrial loci. In cells obtained from fresh tissue sections, 64.5% of alleles successfully amplified to approximately 700000 copies each, and mitochondrial DNA was amplified successfully in all cells. Multiplex PCR amplification and analysis of cells from pre-stored sections yielded significantly poorer results. Sequencing and capillary electrophoresis of WGA products allowed detection of slippage mutations in microsatellites (MS), and point mutations in P53.

Conclusion: Comprehensive genomic analysis of single cells from stained tissue sections opens new research opportunities for cell lineage and depth analyses, genome-wide mutation surveys, and other single cell assays.

Show MeSH
Single cell genome amplification procedure. (A) Tissues of interest are excised and snap frozen in liquid nitrogen. After sectioning, staining, and mounting on a polyethylene membrane coated slide, a cell of interest is laser micro-dissected and catapulted into an adhesive cap of a micro-centrifuge tube. The cell is then subject to DNA extraction and WGA in a protected chamber, minimizing the chance for contamination. Aliquots of the WGA products are amplified by multiple PCRs with specific primers for analysis of multiple genomic loci. (B) Serial photographs taken during laser micro-dissection and catapulting of a single cell. The left panel shows a stained tissue section under low magnification. A portion of the tissue section is viewed under high magnification before (1) and after (2) micro-dissection, and after catapulting (3) of the single cell (bar = 6 μm). Inspection of the adhesive cap under low magnification (4) reveals a catapulted single cell.
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Figure 1: Single cell genome amplification procedure. (A) Tissues of interest are excised and snap frozen in liquid nitrogen. After sectioning, staining, and mounting on a polyethylene membrane coated slide, a cell of interest is laser micro-dissected and catapulted into an adhesive cap of a micro-centrifuge tube. The cell is then subject to DNA extraction and WGA in a protected chamber, minimizing the chance for contamination. Aliquots of the WGA products are amplified by multiple PCRs with specific primers for analysis of multiple genomic loci. (B) Serial photographs taken during laser micro-dissection and catapulting of a single cell. The left panel shows a stained tissue section under low magnification. A portion of the tissue section is viewed under high magnification before (1) and after (2) micro-dissection, and after catapulting (3) of the single cell (bar = 6 μm). Inspection of the adhesive cap under low magnification (4) reveals a catapulted single cell.

Mentions: The procedure for amplifying genomes of laser micro-dissected single cells from frozen tissue sections is outlined in Figure 1A. (full protocols are presented in the materials and methods section). Tissue freezing, sectioning, and staining are performed using standard protocols. In order to minimize the risk of laser-induced damage to DNA, thin (6 μm) sections are used, and micro-dissection is performed using the lowest possible energy level (determined empirically for each section, see materials and methods). Laser ablation paths are typically less than 1 μm in thickness, allowing for isolation of intact cell nuclei with contiguous cytoplasm (Figure 1B). DNA extraction and WGA are performed using the GenomiPhi DNA amplification kit reagents and protocol, with modifications. In order to reduce the risk of contamination from extraneous DNA sources, which is a major concern in single-cell procedures, stringent precautions are employed. These include prior treatment of work surfaces with DNA-destroying agents, use of dedicated pipettes and other instruments, performing DNA extraction and WGA in a restricted chamber with a UV lamp, and the use of a blank control from each tissue section in subsequent WGA and PCR reactions. Aliquots of amplified products can be used directly (without purification) as DNA templates in PCRs for analyses of specific genomic loci.


Amplification of multiple genomic loci from single cells isolated by laser micro-dissection of tissues.

Frumkin D, Wasserstrom A, Itzkovitz S, Harmelin A, Rechavi G, Shapiro E - BMC Biotechnol. (2008)

Single cell genome amplification procedure. (A) Tissues of interest are excised and snap frozen in liquid nitrogen. After sectioning, staining, and mounting on a polyethylene membrane coated slide, a cell of interest is laser micro-dissected and catapulted into an adhesive cap of a micro-centrifuge tube. The cell is then subject to DNA extraction and WGA in a protected chamber, minimizing the chance for contamination. Aliquots of the WGA products are amplified by multiple PCRs with specific primers for analysis of multiple genomic loci. (B) Serial photographs taken during laser micro-dissection and catapulting of a single cell. The left panel shows a stained tissue section under low magnification. A portion of the tissue section is viewed under high magnification before (1) and after (2) micro-dissection, and after catapulting (3) of the single cell (bar = 6 μm). Inspection of the adhesive cap under low magnification (4) reveals a catapulted single cell.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2266725&req=5

Figure 1: Single cell genome amplification procedure. (A) Tissues of interest are excised and snap frozen in liquid nitrogen. After sectioning, staining, and mounting on a polyethylene membrane coated slide, a cell of interest is laser micro-dissected and catapulted into an adhesive cap of a micro-centrifuge tube. The cell is then subject to DNA extraction and WGA in a protected chamber, minimizing the chance for contamination. Aliquots of the WGA products are amplified by multiple PCRs with specific primers for analysis of multiple genomic loci. (B) Serial photographs taken during laser micro-dissection and catapulting of a single cell. The left panel shows a stained tissue section under low magnification. A portion of the tissue section is viewed under high magnification before (1) and after (2) micro-dissection, and after catapulting (3) of the single cell (bar = 6 μm). Inspection of the adhesive cap under low magnification (4) reveals a catapulted single cell.
Mentions: The procedure for amplifying genomes of laser micro-dissected single cells from frozen tissue sections is outlined in Figure 1A. (full protocols are presented in the materials and methods section). Tissue freezing, sectioning, and staining are performed using standard protocols. In order to minimize the risk of laser-induced damage to DNA, thin (6 μm) sections are used, and micro-dissection is performed using the lowest possible energy level (determined empirically for each section, see materials and methods). Laser ablation paths are typically less than 1 μm in thickness, allowing for isolation of intact cell nuclei with contiguous cytoplasm (Figure 1B). DNA extraction and WGA are performed using the GenomiPhi DNA amplification kit reagents and protocol, with modifications. In order to reduce the risk of contamination from extraneous DNA sources, which is a major concern in single-cell procedures, stringent precautions are employed. These include prior treatment of work surfaces with DNA-destroying agents, use of dedicated pipettes and other instruments, performing DNA extraction and WGA in a restricted chamber with a UV lamp, and the use of a blank control from each tissue section in subsequent WGA and PCR reactions. Aliquots of amplified products can be used directly (without purification) as DNA templates in PCRs for analyses of specific genomic loci.

Bottom Line: In cells obtained from fresh tissue sections, 64.5% of alleles successfully amplified to approximately 700000 copies each, and mitochondrial DNA was amplified successfully in all cells.Multiplex PCR amplification and analysis of cells from pre-stored sections yielded significantly poorer results.Comprehensive genomic analysis of single cells from stained tissue sections opens new research opportunities for cell lineage and depth analyses, genome-wide mutation surveys, and other single cell assays.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel. dan.frumkin@weizmann.ac.il

ABSTRACT

Background: Whole genome amplification (WGA) and laser assisted micro-dissection represent two recently developed technologies that can greatly advance biological and medical research. WGA allows the analysis of multiple genomic loci from a single genome and has been performed on single cells from cell suspensions and from enzymatically-digested tissues. Laser micro-dissection makes it possible to isolate specific single cells from heterogeneous tissues.

Results: Here we applied for the first time WGA on laser micro-dissected single cells from stained tissue sections, and developed a protocol for sequentially performing the two procedures. The combined procedure allows correlating the cell's genome with its natural morphology and precise anatomical position. From each cell we amplified 122 genomic and mitochondrial loci. In cells obtained from fresh tissue sections, 64.5% of alleles successfully amplified to approximately 700000 copies each, and mitochondrial DNA was amplified successfully in all cells. Multiplex PCR amplification and analysis of cells from pre-stored sections yielded significantly poorer results. Sequencing and capillary electrophoresis of WGA products allowed detection of slippage mutations in microsatellites (MS), and point mutations in P53.

Conclusion: Comprehensive genomic analysis of single cells from stained tissue sections opens new research opportunities for cell lineage and depth analyses, genome-wide mutation surveys, and other single cell assays.

Show MeSH