An Efficient Probe-Based Quantitative PCR Assay Targeting Human-Specific DNA in ST6GALNAC3 for the Quantification of Human Cells in Preclinical Animal Models

Jinfeng Ren; Ke Liu; Lang Hu; Ruoning Yang; Yuting Liu; Siyu Wang; Xinzhu Chen; Shuli Zhao; Luyao Jing; Tiantian Liu; Bin Hu; Xuefeng Zhang; Hui Wang; Hui Li

doi:10.1007/s12033-024-01115-8

An Efficient Probe-Based Quantitative PCR Assay Targeting Human-Specific DNA in ST6GALNAC3 for the Quantification of Human Cells in Preclinical Animal Models

Mol Biotechnol. 2024 Mar 8. doi: 10.1007/s12033-024-01115-8. Online ahead of print.

Authors

Jinfeng Ren^#^{1

2}, Ke Liu^#³, Lang Hu^#^{1

2}, Ruoning Yang^#⁴, Yuting Liu^{1

2}, Siyu Wang^{1

2}, Xinzhu Chen^{1

2}, Shuli Zhao⁵, Luyao Jing⁴, Tiantian Liu⁴, Bin Hu⁴, Xuefeng Zhang⁶, Hui Wang^{7

8}, Hui Li^{9

10

11

12}

Affiliations

¹ Jiangsu Key Laboratory of Immunity and Metabolism, Department of Pathogenic Biology and Immunology, Xuzhou Medical University, Xuzhou, 221004, Jiangsu, China.
² National Experimental Demonstration Center for Basic Medicine Education, Xuzhou Medical University, Xuzhou, 221004, Jiangsu, China.
³ Department of Gastroenterology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221000, Jiangsu, China.
⁴ Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical University, Xuzhou, 221004, Jiangsu, China.
⁵ General Clinical Research Center, Nanjing First Hospital, Nanjing Medical University, Nanjing, China.
⁶ Jiangsu Tripod Preclinical Research Laboratories Inc, Nanjing, China.
⁷ Jiangsu Key Laboratory of Immunity and Metabolism, Department of Pathogenic Biology and Immunology, Xuzhou Medical University, Xuzhou, 221004, Jiangsu, China. hui.wang@xzhmu.edu.cn.
⁸ National Experimental Demonstration Center for Basic Medicine Education, Xuzhou Medical University, Xuzhou, 221004, Jiangsu, China. hui.wang@xzhmu.edu.cn.
⁹ Jiangsu Key Laboratory of Immunity and Metabolism, Department of Pathogenic Biology and Immunology, Xuzhou Medical University, Xuzhou, 221004, Jiangsu, China. lihuishenghua@xzhmu.edu.cn.
¹⁰ National Experimental Demonstration Center for Basic Medicine Education, Xuzhou Medical University, Xuzhou, 221004, Jiangsu, China. lihuishenghua@xzhmu.edu.cn.
¹¹ Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical University, Xuzhou, 221004, Jiangsu, China. lihuishenghua@xzhmu.edu.cn.
¹² Department of Pathogenic Biology and Immunology, Xuzhou Medical University, 209 Tongshan Road, Xuzhou, 221004, China. lihuishenghua@xzhmu.edu.cn.

^# Contributed equally.

PMID: 38456963
DOI: 10.1007/s12033-024-01115-8

Abstract

Precise quantification of human cells in preclinical animal models by a sensitive and specific approach is warranted. The probe-based quantitative PCR (qPCR) assay as a sensitive and swift approach is suitable for the quantification of human cells by targeting human-specific DNA sequences. In this study, we developed an efficient qPCR assay targeting human-specific DNA in ST6GALNAC3 (termed ST6GAL-qPCR) for the quantification of human cells in preclinical animal models. ST6GAL-qPCR probe was synthesized with FAM and non-fluorescent quencher-minor groove binder conjugated to the 5' and 3' end of the probe, respectively. Genomic DNA from human, rhesus monkeys, cynomolgus monkeys, New Zealand White rabbits, SD rats, C57BL/6, and BALB/c mice were utilized for analyzing the specificity and sensitivity of the ST6GAL-qPCR assay. The ST6GAL-qPCR assay targeted human-specific DNA was cloned to pUCM-T vector and released by EcoR I/Hind III digestion for generating a calibration curve. Cell mixing experiment was performed to validate the ST6GAL-qPCR assay by analysis of 0.1%, 0.01%, and 0.001% of human leukocytes mixed with murine thymocytes. The ST6GAL-qPCR assay detected human DNA rather than DNA from the tested animal species. The amplification efficiency of the ST6GAL-qPCR assay was 93% and the linearity of calibration curve was R² = 0.999. The ST6GAL-qPCR assay detected as low as 5 copies of human-specific DNA and is efficient to specially amplify as low as 30-pg human DNA in the presence of 1 μg of DNA from the tested species, respectively. The ST6GAL-qPCR assay was able to quantify as low as 0.01% of human leukocytes within murine thymocytes. This ST6GAL-qPCR assay can be used as an efficient approach for the quantification of human cells in preclinical animal models.

Keywords: ST6GALNAC3; Preclinical animal models; Quantitative PCR.

Abstract

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