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Validation of droplet digital PCR for cytokeratin 19 mRNA detection in canine peripheral blood and mammary gland

In humans, peripheral blood cytokeratin 19 (CK19) mRNA-positive circulating tumor cells (CTCs) was utilized to identify early-stage breast cancer patients with micrometastatic disease who are at risk for disease progression and monitor treatment response in patients with advanced disease. To our kno...

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Autores principales: Tanvetthayanont, Potsawat, Yata, Teerapong, Boonnil, Jiranun, Temisak, Sasithon, Ponglowhapan, Suppawiwat
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9365843/
https://www.ncbi.nlm.nih.gov/pubmed/35948591
http://dx.doi.org/10.1038/s41598-022-17493-5
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author Tanvetthayanont, Potsawat
Yata, Teerapong
Boonnil, Jiranun
Temisak, Sasithon
Ponglowhapan, Suppawiwat
author_facet Tanvetthayanont, Potsawat
Yata, Teerapong
Boonnil, Jiranun
Temisak, Sasithon
Ponglowhapan, Suppawiwat
author_sort Tanvetthayanont, Potsawat
collection PubMed
description In humans, peripheral blood cytokeratin 19 (CK19) mRNA-positive circulating tumor cells (CTCs) was utilized to identify early-stage breast cancer patients with micrometastatic disease who are at risk for disease progression and monitor treatment response in patients with advanced disease. To our knowledge, there has been little research regarding CK19 in canine mammary tumors (CMTs) using molecular methods. A droplet digital PCR (ddPCR) is proposed as a precise and sensitive quantification of nucleic acid targets. Hence, this study aimed to validate a newly designed assay for CK19 detection in canine blood and mammary tissue, along with the reference gene HPRT, by ddPCR. All primers and probes showed a precise match with the exon region of target genes. The assay exhibited PCR efficacy of 90.4% and 91.0% for CK19 and HPRT amplifications with linearity, respectively. The annealing temperature (T(a)) for duplex ddPCR was 55 °C, providing the highest concentrations of both genes tested by the synthetic plasmid DNA. The limit of detection (LOD) of CK19 and HPRT were 2.16 ± 1.27 and 2.44 ± 1.31 copies/µL, respectively. Finally, the ddPCR assay was validated with canine peripheral blood, non-neoplastic mammary tissues and spiked samples. Our findings provide a new platform for CK19 studies in CMT diagnosis through blood and mammary tissues.
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spelling pubmed-93658432022-08-12 Validation of droplet digital PCR for cytokeratin 19 mRNA detection in canine peripheral blood and mammary gland Tanvetthayanont, Potsawat Yata, Teerapong Boonnil, Jiranun Temisak, Sasithon Ponglowhapan, Suppawiwat Sci Rep Article In humans, peripheral blood cytokeratin 19 (CK19) mRNA-positive circulating tumor cells (CTCs) was utilized to identify early-stage breast cancer patients with micrometastatic disease who are at risk for disease progression and monitor treatment response in patients with advanced disease. To our knowledge, there has been little research regarding CK19 in canine mammary tumors (CMTs) using molecular methods. A droplet digital PCR (ddPCR) is proposed as a precise and sensitive quantification of nucleic acid targets. Hence, this study aimed to validate a newly designed assay for CK19 detection in canine blood and mammary tissue, along with the reference gene HPRT, by ddPCR. All primers and probes showed a precise match with the exon region of target genes. The assay exhibited PCR efficacy of 90.4% and 91.0% for CK19 and HPRT amplifications with linearity, respectively. The annealing temperature (T(a)) for duplex ddPCR was 55 °C, providing the highest concentrations of both genes tested by the synthetic plasmid DNA. The limit of detection (LOD) of CK19 and HPRT were 2.16 ± 1.27 and 2.44 ± 1.31 copies/µL, respectively. Finally, the ddPCR assay was validated with canine peripheral blood, non-neoplastic mammary tissues and spiked samples. Our findings provide a new platform for CK19 studies in CMT diagnosis through blood and mammary tissues. Nature Publishing Group UK 2022-08-10 /pmc/articles/PMC9365843/ /pubmed/35948591 http://dx.doi.org/10.1038/s41598-022-17493-5 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Tanvetthayanont, Potsawat
Yata, Teerapong
Boonnil, Jiranun
Temisak, Sasithon
Ponglowhapan, Suppawiwat
Validation of droplet digital PCR for cytokeratin 19 mRNA detection in canine peripheral blood and mammary gland
title Validation of droplet digital PCR for cytokeratin 19 mRNA detection in canine peripheral blood and mammary gland
title_full Validation of droplet digital PCR for cytokeratin 19 mRNA detection in canine peripheral blood and mammary gland
title_fullStr Validation of droplet digital PCR for cytokeratin 19 mRNA detection in canine peripheral blood and mammary gland
title_full_unstemmed Validation of droplet digital PCR for cytokeratin 19 mRNA detection in canine peripheral blood and mammary gland
title_short Validation of droplet digital PCR for cytokeratin 19 mRNA detection in canine peripheral blood and mammary gland
title_sort validation of droplet digital pcr for cytokeratin 19 mrna detection in canine peripheral blood and mammary gland
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9365843/
https://www.ncbi.nlm.nih.gov/pubmed/35948591
http://dx.doi.org/10.1038/s41598-022-17493-5
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