Dataset Information

Highly sensitive and specific Alu-based quantification of human cells among rodent cells.

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.

SUBMITTER: Funakoshi K

PROVIDER: S-EPMC5643497 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Highly sensitive and specific Alu-based quantification of human cells among rodent cells.

Funakoshi Kodai K Bagheri Mozhdeh M Zhou Ming M Suzuki Ryoji R Abe Hiroshi H Akashi Hideo H

Scientific reports 20171016 1

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 p ...[more]

PMID: 29038571

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Project description:BACKGROUND: Amyloid precursor protein (APP), a key molecule in Alzheimer's disease (AD), is metabolized in two alternative cleavages, generating either the amyloidogenic peptides involved in AD pathology or the soluble form of APP (sAPP?). The level of amyloidogenic peptides in human cerebrospinal fluid (CSF) is considered to be a biomarker of AD, whereas the level of sAPP? in CSF as a biomarker has not been clearly established. sAPP? has neurotrophic and neuroprotective properties. Stimulating its formation and secretion is a promising therapeutic target in AD research. To this end, very sensitive tests for preclinical and clinical research are required. METHODS: The tests are based on homogenous time-resolved fluorescence and require no washing steps. RESULTS: We describe two new rapid and sensitive tests for quantifying mouse and human sAPP?. These 20 ?l-volume tests quantify the levels of: i) endogenous mouse sAPP? in the conditioned medium of mouse neuron primary cultures, as well as in the CSF of wild-type mice, ii) human sAPP? in the CSF of AD mouse models, and iii) human sAPP? in the CSF of AD and non-AD patients. These tests require only 5??l of conditioned medium from 5 × 10(4) mouse primary neurons, 1??l of CSF from wild-type and transgenic mice, and 0.5??l of human CSF. CONCLUSIONS: The high sensitivity of the mouse sAPP? test will allow high-throughput investigations of molecules capable of increasing the secretion of endogenous sAPP? in primary neurons, as well as the in vivo validation of molecules of interest through the quantification of sAPP? in the CSF of treated wild-type mice. Active molecules could then be tested in the AD mouse models by quantifying human sAPP? in the CSF through the progression of the disease. Finally, the human sAPP? test could strengthen the biological diagnosis of AD in large clinical investigations. Taken together, these new tests have a wide field of applications in preclinical and clinical studies.

Project description:BACKGROUND: Coxiella burnetii, the bacterium causing Q fever, is an obligate intracellular biosafety level 3 agent. Detection and quantification of these bacteria with conventional methods is time consuming and dangerous. During the last years, several PCR based diagnostic assays were developed to detect C. burnetii DNA in cell cultures and clinical samples. We developed and evaluated TaqMan-based real-time PCR assays that targeted the singular icd (isocitrate dehydrogenase) gene and the transposase of the IS1111a element present in multiple copies in the C. burnetii genome. RESULTS: To evaluate the precision of the icd and IS1111 real-time PCR assays, we performed different PCR runs with independent DNA dilutions of the C. burnetii Nine Mile RSA493 strain. The results showed very low variability, indicating efficient reproducibility of both assays. Using probit analysis, we determined that the minimal number of genome equivalents per reaction that could be detected with a 95% probability was 10 for the icd marker and 6.5 for the IS marker. Plasmid standards with cloned icd and IS1111 fragments were used to establish standard curves which were linear over a range from 10 to 10(7) starting plasmid copy numbers. We were able to quantify cell numbers of a diluted, heat-inactivated Coxiella isolate with a detection limit of 17 C. burnetii particles per reaction. Real-time PCR targeting both markers was performed with DNA of 75 different C. burnetii isolates originating from all over the world. Using this approach, the number of IS1111 elements in the genome of the Nine Mile strain was determined to be 23, close to 20, the number revealed by genome sequencing. In other isolates, the number of IS1111 elements varied widely (between seven and 110) and seemed to be very high in some isolates. CONCLUSION: We validated TaqMan-based real-time PCR assays targeting the icd and IS1111 markers of C. burnetii. The assays were shown to be specific, highly sensitive and efficiently reproducible. Cell numbers in dilutions of a C. burnetii isolate were reliably quantified. PCR quantification suggested a high variability of the number of IS1111 elements in different C. burnetii isolates, which may be useful for further phylogenetic studies.

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Highly sensitive and specific Alu-based quantification of human cells among rodent cells.

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Highly sensitive and specific Alu-based quantification of human cells among rodent cells.

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