A systematic evaluation of stool DNA preparation protocols for colorectal cancer screening via analysis of DNA methylation biomarkers

Abstract

ObjectivesColorectal 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.MethodsWe 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.ResultsThe 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.ConclusionsThrough 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.

Systematic Identification of UBE2C As a Prognostic Biomarker and Correlated with Immunosuppression and Invasiveness in Glioma

“…Currently, non-coding RNA, circulating exosome factors and cerebrospinal fluid can be the scope of seeking glioma biomarkers [7, 34, 36–38]. However, this study suggests that in addition to circulatory source factor expression differences and prognostic analysis, systematic analysis and evaluation based on markers are also key to validate their reliability [39, 40], such as assessing the intrinsic and extrinsic association and risk of marker genes, which are also needed. Therefore, a relatively typical marker - UBE2C was selected based on systemic intrinsic factors such as differential expression genes, risk genes, cell cycle and DNA replication in glioma.…”

UBE2C is associated with Prognosis of Immunosuppression and Cell Invasiveness in Glioma

“…Currently, non-coding RNA, circulating exosome factors and cerebrospinal uid can be the scope of seeking glioma biomarkers [7,34,[36][37][38]. However, this study suggests that in addition to circulatory source factor expression differences and prognostic analysis, systematic analysis and evaluation based on markers are also key to validate their reliability [39,40], such as assessing the intrinsic and extrinsic association and risk of marker genes, which are also needed. Therefore, a relatively typical marker -UBE2C was selected based on systemic intrinsic factors such as differential expression genes, risk genes, cell cycle and DNA replication in glioma.…”

Use of Omics Technologies for the Detection of Colorectal Cancer Biomarkers

“…Another similar report performed a methylation analysis using MethyLight qPCR and droplet digital PCR, reporting elevated CpG islands methylation in two genes: GRIA4 and VIPR2 [60]. Two additional DNA methylation markers analysed with multiplex quantitative PCR assay, SDC2 and NDRG4, were found in another study, providing a solid foundation for multi-target DNA biomarker analysis in stool samples for CRC screening [61]. Previously, other authors reported SDC2 methylation as a good candidate for potential non-invasive diagnostic tool for early detection of CRC [62].…”