Abstract
Objectives
Colorectal cancer (CRC) screening using stool samples is now in routine use where tumor DNA methylation analysis for leading markers such as NDRG4 and SDC2 is an integral part of the test. However, processing stool samples for reproducible and efficient extraction of human genomic DNA remains a bottleneck for further research into better biomarkers and assays.
Methods
We systematically evaluated several factors involved in the processing of stool samples and extraction of DNA. These factors include: stool processing (solid and homogenized samples), preparation of DNA from supernatant and pellets, and DNA extraction with column and magnetic beads-based methods. Furthermore, SDC2 and NDRG4 methylation levels were used to evaluate the clinical performance of the optimal protocol.
Results
The yield of total and human genomic DNA (hgDNA) was not reproducible when solid stool scraping is used, possibly due to sampling variations. More reproducible results were obtained from homogenized stool samples. Magnetic beads-based DNA extraction using the supernatant from the homogenized stool was chosen for further analysis due to better reproducibility, higher hgDNA yield, lower non-hgDNA background, and the potential for automation. With this protocol, a combination of SDC2 and NDRG4 methylation signals with a linear regression model achieved a sensitivity and specificity of 81.82 and 93.75%, respectively.
Conclusions
Through the systematic evaluation of different stool processing and DNA extraction methods, we established a reproducible protocol for analyzing tumor DNA methylation markers in stool samples for colorectal cancer screening.
Funding source: National Natural Sciences Foundation of China
Award Identifier / Grant number: 81672922
Funding source: Medical Health Science and Technology Project of Zhejiang Provincial Health Commission
Award Identifier / Grant number: 2017174160
Funding source: Wenzhou Science and Technology Bureau
Award Identifier / Grant number: Y20160044
Award Identifier / Grant number: 2018ZY005
Funding source: High-Level Innovation Team of Universities in Zhejiang Province
Award Identifier / Grant number: 604090352/610
Funding source: Innovation Discipline of Zhejiang Province in Nucleic Acid Molecular Diagnostics
Award Identifier / Grant number: 437201702G
Funding source: Key Discipline of Zhejiang Province in Medical Technology
Award Identifier / Grant number: 437601607
Acknowledgments
We would like to thank all the patients who participated in the studay and all the support from the doctors and nurses for the collection of samples.
Research funding: This work was supported by the National Natural Sciences Foundation of China (CD: No. 81672922), Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (CX: No. 2017174160), Wenzhou Science and Technology Bureau (CD: No. 2018ZY005; CX: No. Y20160044), High-Level Innovation Team of Universities in Zhejiang Province (CD, No. 604090352/610), Innovation Discipline of Zhejiang Province in Nucleic Acid Molecular Diagnostics (CD: No. 437201702G) and Key Discipline of Zhejiang Province in Medical Technology (Fist Class, Category A) (CD: No. 437601607).
Author contributions: Shengnan Jin, Chang Xu and Chunming Ding: design of the project, data interpretation, writing of the manuscript; Qian Ye: performance of lab work and interpretation of data, writing of the manuscript; Zhengzheng Zhang, Shiliang Chen, Yourong Wang, Dandan Li, Wen Ma, Yanping Hong, Dewen Zhu, Wenqing Dai, Chengliang Zhang, Weihao Liu and Ying Guo: sample collection and clinical database curation; Zhengquan Yang, Jinlei Li, Ju Luan, Xiaoli Wu, Feizhao Jiang and Zhihai Zheng: patient enrollment. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Competing interests: Authors state no conflict of interest.
Informed consent: Informed consent was obtained from all individuals included in this study.
Ethical approval: The study was approved by the Clinical Research Ethics of the First Affiliated Hospital of Wenzhou Medical University.
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Supplementary material
The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2020-0300).
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