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An efficient qPCR assay for the quantification of human cells in preclinical animal models by targeting human specific DNA in the intron of BRCA1

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Abstract

Background

Precise quantification of grafted human cells in preclinical animal models such as non-human primates, rodents and rabbits is needed for the evaluations of the safety and efficacy of cell therapy. Quantitative PCR (qPCR) as a swift, sensitive and powerful assay is suitable for human cell quantification. However, it is a formidable challenge due to that the genome of non-human primates share more than 95% of similarity as human. Methods: In the present study, we developed a probe-based quantitative PCR (qPCR) assay for the quantification of human cells in preclinical animal models via targeting human specific DNA in the intron of BRCA1 (termed BRCA1-qPCR). The 5′ and 3′ end of BRCA1-qPCR probe was conjugated with FAM and non-fluorescent quencher-minor groove binder (NFQ-MGB), respectively. 1 µg of genomic DNA from human and preclinical animal models including rhesus monkeys, cynomolgus monkeys, New Zealand white rabbits, SD rats, C57BL/6 and BALB/c mice were used for determining the specificity and sensitivity of the BRCA1-qPCR assay. A calibration curve was generated by BRCA1-qPCR analysis of linearized plasmid containing targeted human specific DNA in BRCA1. The BRCA1-qPCR assay was validated by analysis of 0.003%, 0.03% and 0.3% of human leukocytes mixed within murine leukocytes.

Results

The BRCA1-qPCR assay detected human DNA rather than DNA from tested species. The amplification efficiency of the BRCA1-qPCR assay was 95.4% and the linearity of the calibration curve was R2 = 0.9997. The BRCA1-qPCR assay detected as low as 5 copies of human specific DNA and is efficient to specially amplify 30 pg human DNA in the presence of 1 µg of genomic DNA from tested species, respectively. The BRCA1-qPCR assay was able to quantify as low as 0.003% of human cells within murine leukocytes.

Conclusion

The BRCA1-qPCR assay is efficient for the quantification of human cells in preclinical animal models.

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Data availability

All data generated or analyzed during this study are included in this article.

Abbreviations

qPCR:

quantitative PCR

BLAST:

Basic local alignment search tool

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Funding

This work was supported by the National Natural Science Foundation of China (grant no. 82171791) and the Youth Innovation Team Grant and the Starting Grant by Xuzhou Medical University (grant no. D2018009), Xuzhou Technology Program (KC20089), and the Postgraduate Research & Practice Innovation Program of Jiangsu Province, China (KYCX21_2638).

Author information

Author notes

  1. Ke Liu, Lang Hu and Siyu Wang have been contributed equally to this work.

Authors and Affiliations

  1. Department of Gastroenterology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221000, Jiangsu, China

    Ke Liu & Li Li

  2. Jiangsu Key Laboratory of Immunity and Metabolism, Department of Pathogenic Biology and Immunology, Xuzhou Medical University, 209 Tongshan Road, Xuzhou, 221004, Jiangsu, China

    Lang Hu, Siyu Wang, Xinzhu Chen, Yuting Liu, Hui Wang & Hui Li

  3. National Experimental Demonstration Center for Basic Medicine Education, Xuzhou Medical University, Xuzhou, 221004, Jiangsu, China

    Lang Hu, Siyu Wang, Xinzhu Chen, Yuting Liu, Hui Wang & Hui Li

  4. General Clinical Research Center, Nanjing First Hospital, Nanjing Medical University, Nanjing, China

    Shuli Zhao

  5. Department of Biotechnology, Xuzhou Medical University, Xuzhou, 221004, Jiangsu, China

    Hui Li

Authors
  1. Ke Liu
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Contributions

KL, LH, SW, XC and YL performed the experiments. LH and KL performed data analysis. SZ collected human samples. LH, LL and HW conceived and supervised the study. LH prepared the figures and wrote the manuscript with input from HW and LL.

Corresponding authors

Correspondence to Li Li or Hui Li.

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Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Ethics approval

All animal studies were performed in accordance with the protocol approved by the Animal Experimental Ethics Committee of Xuzhou Medical University(Approval ID: 202211S011). The usage of blood samples was approved by the ethics committee of Nanjing First Hospital (Approval ID: KY20220516-04).

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Informed consent was obtained from all individual participants included in the study.

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11033_2023_8853_MOESM1_ESM.png

Figure S1. Analysis of human genomic DNA by the BRCA1-qPCR assay. The representative amplification plot of the BRCA1-qPCRanalysis of genomic DNA (1-10 ng) of 48 individuals (PNG 161.1 kb)

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Liu, K., Hu, L., Wang, S. et al. An efficient qPCR assay for the quantification of human cells in preclinical animal models by targeting human specific DNA in the intron of BRCA1. Mol Biol Rep 50, 9229–9237 (2023). https://doi.org/10.1007/s11033-023-08853-z

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