Abstract
Lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus (EBV) serve as an unlimited resource of human genomic DNA. The protocol that is widely used to establish LCLs involves peripheral blood mononuclear cell isolation by density gradient centrifugation, however, that method requires as much as 5 ml of peripheral blood. In this study, in order to provide a more simple and efficient method for the generation of LCLs, we developed a new protocol using hemolytic reaction to enrich white blood cells for EBV transformation and found that the hemolytic protocol successfully generated LCLs from a small volume (i.e., 0.1 ml) of peripheral blood. To assess the quality of genomic DNA extracted from LCLs established by the hemolytic protocol (LCL-hemolytic), we performed single nucleotide polymorphism (SNP) microarray genotyping using the GeneChip® 100 K Array Set (Affymetrix, Inc.). The concordances of the SNP genotyping resulting from genomic DNA from LCL-hemolytic (99.92%) were found to be as good as the technical replicate (99.90%), and Kappa statistics results confirmed the reliability. The findings of this study reveal that the hemolytic protocol is a simple and reliable method for the generation of LCLs, even from a small volume of peripheral blood.
Highlights
A wide variety of commercially available reagents that allow researchers to separate PBMCs from the different layers of the other components of blood based on each density[13,14,15]
In order to evaluate the influence of EBV infection and transformation to the genomic DNA of LCLs, single nucleotide polymorphism (SNP) genotype data obtained from genomic DNA derived from peripheral blood and that obtained from genomic DNA derived from LCL-hemolytic were compared (Fig. 1c)
Our findings showed that the concordance rate of the genotype data between the genomic DNA derived from LCLs and its peripheral blood turned out to be sufficiently high (>99.80%) in light of the concordance rate of the technical replicates (99.90%) (Table 5)
Summary
A wide variety of commercially available reagents that allow researchers to separate PBMCs from the different layers of the other components of blood based on each density[13,14,15]. The gradient protocol typically requires 5 ml or more of peripheral blood in order for it to be overlaid onto the gradient-making reagent[16,17,18]. Post centrifugation, the method required to collect the interface layer, which contains PBMCs, is complex. In order to assess and confirm that the quality of the genomic DNA extracted from LCLs established by this novel method using hemolytic reaction (LCL-hemolytic) is as good as genomic DNA extracted from peripheral blood and genomic DNA extracted from LCLs established by the conventional method using density gradient centrifugation (LCL-gradient), we performed single nucleotide polymorphism (SNP) microarray genotyping using the. ® GeneChip 100 K Array Set (Affymetrix, Inc., Santa Clara, CA), and compared the concordance of genotyping results using each of the genomic DNAs
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