Abstract
Alu elements are primate-specific short interspersed elements (SINEs), over 1 million copies of which are present in the human genome; thus, Alu elements are useful targets for detecting human cells. However, previous Alu-based techniques for detecting human genomic DNA do not reach the theoretical limits of sensitivity and specificity. In this study, we developed a highly sensitive and specific Alu-based real-time PCR method for discriminating human cells from rodent cells, using a primer and probe set carefully designed to avoid possible cross-reactions with rodent genomes. From 100 ng of mixed human and rodent genomes, 1 fg of human genome, equivalent to 1 human cell in 100 million rodent cells, was detectable. Furthermore, in vivo mouse subrenal capsule xenotransplantation assays revealed that 10 human cells per mouse organ were detectable. In addition, after intravenous injection of human mesenchymal stem cells into NOD/SCID mice via tail vein, the biodistribution of human cells was trackable in the mouse lungs and kidneys for at least 1 week. Our findings indicate that our primer and probe set is applicable for the quantitative detection of tiny amounts of human cells, such as xenotransplanted human cancer or stem cells, in rodents.
Highlights
The sensitive and specific quantification of human DNA using real-time quantitative PCR has played important roles in various fields, such as cancer diagnostics, cancer and stem cell research, and forensic science[1,2,3]
As a proof of principle, we aimed to demonstrate that our Alu-quantitative PCR (qPCR) system was capable of discriminating 1 fg of human genomic material among 100 ng of the rodent genome, which is equivalent to 1 human cell among 100 million rodent cells
As it was necessary to avoid sequences that were extremely homologous to rodent genomes when designing specific primers and probe for the Alu model sequence, we introduced the following constraints as design criteria: i) the primer or probe should not contain more than two 19-nt subsequences that perfectly match the genomic sequences of a specific rodent species, i.e. mouse, rat, or guinea pig; and ii) the primer or probe should be 20 nt or longer
Summary
The sensitive and specific quantification of human DNA using real-time quantitative PCR (qPCR) has played important roles in various fields, such as cancer diagnostics, cancer and stem cell research, and forensic science[1,2,3]. There are over 1 million Alu copies in the human genome, accounting for over 10% of the entire genome[12] Because of their species specificity, small size, and extraordinarily high copy number, Alu elements are ideal targets for qPCR aimed at detecting human cells among the cells derived from other animal species[13,14,15,16,17,18,19,20,21,22,23,24]. In the qPCR system is as low as 3 copies[27], Alu elements should be detectable in this amount of human genomic DNA. Alu-qPCR could unavoidably elicit non-specific reactions resulting in a high background signal All of these possibilities could seriously affect the sensitivity and specificity of Alu-qPCR. To our knowledge, no previous study has adequately addressed these issues
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