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Isolation of salivary cell-free DNA for cancer detection

Saliva is an emerging source of disease biomarkers, particularly for cancers of the head and neck. Although analysis of cell-free DNA (cfDNA) in saliva holds promise as a liquid biopsy for cancer detection, currently there are no standardized methodologies for the collection and isolation of saliva...

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Autores principales: Brooks, Patricia J., Malkin, Ethan Z., De Michino, Steven, Bratman, Scott V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10153704/
https://www.ncbi.nlm.nih.gov/pubmed/37130100
http://dx.doi.org/10.1371/journal.pone.0285214
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author Brooks, Patricia J.
Malkin, Ethan Z.
De Michino, Steven
Bratman, Scott V.
author_facet Brooks, Patricia J.
Malkin, Ethan Z.
De Michino, Steven
Bratman, Scott V.
author_sort Brooks, Patricia J.
collection PubMed
description Saliva is an emerging source of disease biomarkers, particularly for cancers of the head and neck. Although analysis of cell-free DNA (cfDNA) in saliva holds promise as a liquid biopsy for cancer detection, currently there are no standardized methodologies for the collection and isolation of saliva for the purposes of studying DNA. Here, we evaluated various saliva collection receptacles and DNA purification techniques, comparing DNA quantity, fragment size, source, and stability. Then, using our optimized techniques, we tested the ability to detect human papillomavirus (HPV) DNA– a bona fide cancer biomarker in a subset of head and neck cancers– from patient saliva samples. For saliva collection, we found that the Oragene OG-600 receptacle yielded the highest concentration of total salivary DNA as well as short fragments <300 bp corresponding to mononucleosomal cell-free DNA. Moreover, these short fragments were stabilized beyond 48 hours after collection in contrast to other saliva collection receptacles. For DNA purification from saliva, the QIAamp Circulating Nucleic Acid kit yielded the highest concentration of mononucleosome-sized DNA fragments. Freeze-thaw of saliva samples did not affect DNA yield or fragment size distribution. Salivary DNA isolated from the OG-600 receptacle was found to be composed of both single and double-stranded DNA, including mitochondrial and microbial sources. While levels of nuclear DNA were consistent over time, levels of mitochondrial and microbial DNA were more variable and increased 48 hours after collection. Finally, we found that HPV DNA was stable in OG-600 receptacles, was reliably detected within the saliva of patients with HPV-positive head and neck cancer, and was abundant among mononucleosome-sized cell-free DNA fragments. Our studies have defined optimal techniques for isolating DNA from saliva that will contribute to future applications in liquid biopsy-based cancer detection.
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spelling pubmed-101537042023-05-03 Isolation of salivary cell-free DNA for cancer detection Brooks, Patricia J. Malkin, Ethan Z. De Michino, Steven Bratman, Scott V. PLoS One Research Article Saliva is an emerging source of disease biomarkers, particularly for cancers of the head and neck. Although analysis of cell-free DNA (cfDNA) in saliva holds promise as a liquid biopsy for cancer detection, currently there are no standardized methodologies for the collection and isolation of saliva for the purposes of studying DNA. Here, we evaluated various saliva collection receptacles and DNA purification techniques, comparing DNA quantity, fragment size, source, and stability. Then, using our optimized techniques, we tested the ability to detect human papillomavirus (HPV) DNA– a bona fide cancer biomarker in a subset of head and neck cancers– from patient saliva samples. For saliva collection, we found that the Oragene OG-600 receptacle yielded the highest concentration of total salivary DNA as well as short fragments <300 bp corresponding to mononucleosomal cell-free DNA. Moreover, these short fragments were stabilized beyond 48 hours after collection in contrast to other saliva collection receptacles. For DNA purification from saliva, the QIAamp Circulating Nucleic Acid kit yielded the highest concentration of mononucleosome-sized DNA fragments. Freeze-thaw of saliva samples did not affect DNA yield or fragment size distribution. Salivary DNA isolated from the OG-600 receptacle was found to be composed of both single and double-stranded DNA, including mitochondrial and microbial sources. While levels of nuclear DNA were consistent over time, levels of mitochondrial and microbial DNA were more variable and increased 48 hours after collection. Finally, we found that HPV DNA was stable in OG-600 receptacles, was reliably detected within the saliva of patients with HPV-positive head and neck cancer, and was abundant among mononucleosome-sized cell-free DNA fragments. Our studies have defined optimal techniques for isolating DNA from saliva that will contribute to future applications in liquid biopsy-based cancer detection. Public Library of Science 2023-05-02 /pmc/articles/PMC10153704/ /pubmed/37130100 http://dx.doi.org/10.1371/journal.pone.0285214 Text en © 2023 Brooks et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Brooks, Patricia J.
Malkin, Ethan Z.
De Michino, Steven
Bratman, Scott V.
Isolation of salivary cell-free DNA for cancer detection
title Isolation of salivary cell-free DNA for cancer detection
title_full Isolation of salivary cell-free DNA for cancer detection
title_fullStr Isolation of salivary cell-free DNA for cancer detection
title_full_unstemmed Isolation of salivary cell-free DNA for cancer detection
title_short Isolation of salivary cell-free DNA for cancer detection
title_sort isolation of salivary cell-free dna for cancer detection
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10153704/
https://www.ncbi.nlm.nih.gov/pubmed/37130100
http://dx.doi.org/10.1371/journal.pone.0285214
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