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

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.

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.

Figure 2

Amplification of multiple genomic loci from single cells. (A) Tissue section with cells 1–4 marked by white circles (bar = 6 μm). (B) MS locus ABI20 was analyzed by PCR amplification and capillary electrophoresis. Both paternal and maternal alleles are visible in the tail clipping sample (Tail) and in cell 3. Allelic dropout (ADO) can be seen in the short allele of cells 1, 2, and 4. Slippage mutations (arrows) can be seen in the long allele of cells 2 and 4. The negative control (NC) sample shows no amplification. (C) Sequencing of exon 8 from the P53 gene. A point mutation (C => T) can be seen in the 18^th nucleotide from the left (highlighted grey) in cells 1 and 2. In cell 3, both the normal and mutated alleles were amplified, and in cell 4 only a normal allele is visible. (D) Mitochondrial ND3 gene locus was amplified by PCR and run on an agarose gel. Amplification is successful for all cells from a fresh section, but not for all cells from a pre-stored section.