Laser Microdissection and Near Single-Cell Whole Genome Amplification

Area of Science:

• Molecular Biology
• Virology
• Histopathology

Background:

• Precise isolation of virus-infected cells is crucial for genomic analysis.
• Formalin-fixed paraffin-embedded (FFPE) tissues present challenges for nucleic acid recovery.
• Existing methods may struggle with low viral loads and high host DNA background.

Purpose of the Study:

• To present a detailed protocol for laser microdissection (LMD) and near single-cell whole genome amplification (WGA).
• To optimize viral genome recovery and sequencing from FFPE tissues.
• To reduce host genomic interference and enhance pathogen detection.

Main Methods:

• Laser microdissection (LMD) for targeted cell isolation.
• Near single-cell whole genome amplification (WGA) using the SeqPlex DNA Amplification Kit.
• DNA extraction utilizing the PicoPure DNA Extraction Kit from FFPE tissue sections.

Main Results:

• Successful isolation of minimal numbers of virus-infected cells (near single-cell resolution).
• Generation of whole viral genomes with significantly reduced host DNA interference.
• High-quality nucleic acid recovery suitable for downstream next-generation sequencing (NGS) workflows.

Conclusions:

• The integrated LMD and WGA protocol enables precise recovery of viral genomes from FFPE tissues.
• This approach enhances the detection of pathogen-specific signals for NGS.
• The protocol is optimized for challenging FFPE samples, improving viral diagnostics.