title: Ultrahigh-throughput approach for analyzing single-cell genomic damage with an agarose-based microfluidic comet array.
abstract: Genomic DNA damage was generally identified with a "comet assay" but limited by low throughput and poor reproducibility. Here we demonstrated an ultrahigh-throughput approach with a microfluidic chip to simultaneously interrogate DNA damage conditions of up to 10,000 individual cells (approximately 100-fold in throughput over the conventional method) with better reproducibility. For experiment, agarose was chosen as the chip fabrication material, which would further act as an electrophoretic sieving matrix for DNA fragments separation. Cancer cells (HeLa or HepG2) were lined up in parallel microchannels by capillary effect to form a dense array of single cells. After treatment with different doses of hydrogen peroxide, individual cells were then lysed for subsequent single-cell gel electrophoresis in the direction vertical to microchannel and fluorescence detection. Through morphological analysis and fluorescent measurement of comet-shaped DNA, the damage conditions of individual cells could be quantified. DNA repair capacity was further evaluated to validate the reliability of this method. It indicated that the agarose-based microfluidic comet array electrophoresis was simple, highly reproducible, and of high throughput, providing a new method for highly efficient single-cell genomic analysis.
authors.affiliation: Britton Chance Center for Biomedical Photonics at Wuhan National Laboratory for Optoelectronics, Systems Biology Theme, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.
authors.forename: Yiwei
authors.initials: Y
authors.lastname: Li
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Feng
authors.forename: Wei
authors.initials: W
authors.lastname: Du
authors.forename: Ying
authors.initials: Y
authors.lastname: Li
authors.forename: Bi-Feng
authors.initials: BF
authors.lastname: Liu
mesh_terms: D020552:Comet Assay; D004247:DNA / Q000032:analysis*; D004249:DNA Damage; D004260:DNA Repair; D004587:Electrophoresis, Agar Gel; D006367:HeLa Cells; D056945:Hep G2 Cells; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012685:Sepharose; D059010:Single-Cell Analysis / Q000295:instrumentation / Q000379:methods*; D012984:Software
chemical_list: D004247:DNA; D012685:Sepharose; D006861:Hydrogen Peroxide