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Digital PCR quantification of DNA, RNA and extracellular microRNA of mouse oocytes

Joan Xiaohui Yang, Xin Yuan Zhao, Dexi Bi, Qing Wei, Citra Mattar, Joy Yan Ling Pang, Yie Hou Lee
doi: https://doi.org/10.1101/2021.06.03.446991
Joan Xiaohui Yang
1Division of Obstetrics & Gynaecology, KK Women’s and Children’s Hospital, Singapore, Singapore
2Translational ‘Omics and Biomarkers Group, KK Research Centre, KK Women’s and Children’s Hospital, Singapore, Singapore
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Xin Yuan Zhao
3Chemical Engineering & Food Technology, Singapore Institute of Technology, Singapore, Singapore
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Dexi Bi
4Department of Pathology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China
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Qing Wei
4Department of Pathology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China
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Citra Mattar
5Department of Obstetrics & Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
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Joy Yan Ling Pang
3Chemical Engineering & Food Technology, Singapore Institute of Technology, Singapore, Singapore
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Yie Hou Lee
2Translational ‘Omics and Biomarkers Group, KK Research Centre, KK Women’s and Children’s Hospital, Singapore, Singapore
6Obstetrics & Gynaecology Academic Clinical Program, Duke-NUS Medical School, Singapore, Singapore
7Critical Analytics in Manufacturing Precision Medicine, Singapore-MIT Alliance for Research and Technology, Singapore
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  • For correspondence: yiehou.lee@smart.mit.edu
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ABSTRACT

Despite numerous advances in in vitro fertilization (IVF) techniques since its first success in 1978, almost half of the patients treated remain childless. The multifactorial nature of IVF treatment means that success is dependent on variables, including the quality of oocytes. Therefore, new technologies are needed to objectively and quantitatively examine how each oocyte can be selected or optimized to achieve for the best possible outcomes for patients. Here, we report an optimized digital polymerase chain reaction (dPCR) for direct absolute quantification of nucleic acids within 3.5 h without the need for sample extraction or purification. Using individual oocytes, the developed method demonstrated absolute quantification with a linear dynamic range of 0.65 – 33 copies/µL (r2=0.999), high accuracy and excellent reproducibility of <10% relative standard deviation. The method then identified the variable expression of Gapdh (0.72-16.95 copies/oocyte), Hprt1 (1.05-19.05 copies/oocyte) and ATPase 6, (5.55-32358.15 copies/oocyte) in ovaries even from the same mouse. Finally, dPCR was used to validate extracellular microRNAs from oocytes incubated with a toxic unsaturated very-long chained ceramide. This study therefore shows the feasibility of dPCR for the rapid and sensitive absolute quantification of DNA/RNA and extracellular miRNA for the study of oocytes.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Funding: SingHealth Foundation (SHF/FG560P/2014) and National Medical Research Council, Singapore (NMRC/BNIG/2033/2015).

  • Disclosure summary: All authors have nothing to disclose.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted June 04, 2021.
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Digital PCR quantification of DNA, RNA and extracellular microRNA of mouse oocytes
Joan Xiaohui Yang, Xin Yuan Zhao, Dexi Bi, Qing Wei, Citra Mattar, Joy Yan Ling Pang, Yie Hou Lee
bioRxiv 2021.06.03.446991; doi: https://doi.org/10.1101/2021.06.03.446991
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Digital PCR quantification of DNA, RNA and extracellular microRNA of mouse oocytes
Joan Xiaohui Yang, Xin Yuan Zhao, Dexi Bi, Qing Wei, Citra Mattar, Joy Yan Ling Pang, Yie Hou Lee
bioRxiv 2021.06.03.446991; doi: https://doi.org/10.1101/2021.06.03.446991

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