Analysis of Copy-Number Alterations in Single Cells Using Microarray-Based Comparative Genomic Hybridization (aCGH)

Area of Science:

• Genomics
• Molecular Biology
• Cell Biology

Background:

• Array comparative genomic hybridization (aCGH) is a powerful technique for detecting genomic copy number variations.
• Analyzing single cells presents unique challenges due to limited DNA quantity.
• Existing methods often require substantial cell numbers, limiting single-cell applications.

Purpose of the Study:

• To establish a comprehensive workflow for performing aCGH on single cells.
• To optimize protocols for DNA isolation, whole-genome amplification, and aCGH analysis from single cells.
• To provide a detailed guide for researchers interested in single-cell genomic studies.

Main Methods:

• Isolation and preparation of single peripheral blood mononuclear cells (PBMCs) and cell lines (GM14667, GM05423) for reference DNA.
• Micromanipulation techniques for handling single cells.
• Whole-genome amplification (WGA) using adapter-linker PCR (Ampli1 WGA Kit) and a nonlinear WGA method (PicoPLEX WGA Kit).
• Reamplification of WGA products for enhanced aCGH.
• aCGH using 4 × 180k oligonucleotide microarrays.

Main Results:

• Demonstration of a reproducible workflow for single-cell aCGH.
• Successful whole-genome amplification from single cells using two distinct WGA kits.
• aCGH successfully performed on amplified single-cell DNA, enabling detection of genomic alterations.
• Validation of protocols for reference DNA preparation and cell manipulation.

Conclusions:

• The described workflow enables reliable aCGH analysis at the single-cell level.
• This methodology facilitates the study of genomic heterogeneity within cell populations.
• The protocol provides a valuable resource for advancing single-cell genomics research.