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Development of a New Monochrome Multiplex qPCR Method for Relative Telomere Length Measurement in Cancer

Excess telomere shortening has been observed in most cancer cells. The telomere quantitative polymerase chain reaction (qPCR) assay has become an important tool for epidemiological studies examining the effects of aging, stress, and other factors on the length of telomeres. Current telomere qPCR met...

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Autores principales: Dahlgren, Paige N., Bishop, Kanokwan, Dey, Shatovisha, Herbert, Brittney-Shea, Tanaka, Hiromi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Neoplasia Press 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915991/
https://www.ncbi.nlm.nih.gov/pubmed/29573637
http://dx.doi.org/10.1016/j.neo.2018.02.007
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author Dahlgren, Paige N.
Bishop, Kanokwan
Dey, Shatovisha
Herbert, Brittney-Shea
Tanaka, Hiromi
author_facet Dahlgren, Paige N.
Bishop, Kanokwan
Dey, Shatovisha
Herbert, Brittney-Shea
Tanaka, Hiromi
author_sort Dahlgren, Paige N.
collection PubMed
description Excess telomere shortening has been observed in most cancer cells. The telomere quantitative polymerase chain reaction (qPCR) assay has become an important tool for epidemiological studies examining the effects of aging, stress, and other factors on the length of telomeres. Current telomere qPCR methods analyze the relative length of telomeres by amplifying telomere sequence products and normalizing with single-copy gene products. However, the current telomere qPCR does not always reflect absolute telomere length in cancer DNA. Because of genomic instability in cancer cells, we hypothesized that the use of single-copy genes (scg) is less accurate for normalizing data in cancer DNA and that new primer sets are required to better represent relative telomere length in cancer DNA. We first confirmed that cancer cells had a different copy ratio among different scg, implying that DNA is aneuploid. By using the new primer sets that amplify multiple-copy sequences (mcs) throughout the genome, the telomere qPCR results showed that the mcs primers were interchangeable with the scg primers as reference primers in normal DNA. By comparing results from the traditional southern blotting method (as kilobases) and results from monochrome multiplex qPCR using the mcs primers (as T/M ratios), we verified that the T/M ratio is highly correlated with absolute telomere length from the southern blot analysis. Together, the mcs primers were able to represent the telomere lengths accurately in cancer DNA samples. These results would allow for analyses of telomeres within cancerous DNA and the development of new, less invasive diagnostic tools for cancer.
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spelling pubmed-59159912018-04-27 Development of a New Monochrome Multiplex qPCR Method for Relative Telomere Length Measurement in Cancer Dahlgren, Paige N. Bishop, Kanokwan Dey, Shatovisha Herbert, Brittney-Shea Tanaka, Hiromi Neoplasia Original article Excess telomere shortening has been observed in most cancer cells. The telomere quantitative polymerase chain reaction (qPCR) assay has become an important tool for epidemiological studies examining the effects of aging, stress, and other factors on the length of telomeres. Current telomere qPCR methods analyze the relative length of telomeres by amplifying telomere sequence products and normalizing with single-copy gene products. However, the current telomere qPCR does not always reflect absolute telomere length in cancer DNA. Because of genomic instability in cancer cells, we hypothesized that the use of single-copy genes (scg) is less accurate for normalizing data in cancer DNA and that new primer sets are required to better represent relative telomere length in cancer DNA. We first confirmed that cancer cells had a different copy ratio among different scg, implying that DNA is aneuploid. By using the new primer sets that amplify multiple-copy sequences (mcs) throughout the genome, the telomere qPCR results showed that the mcs primers were interchangeable with the scg primers as reference primers in normal DNA. By comparing results from the traditional southern blotting method (as kilobases) and results from monochrome multiplex qPCR using the mcs primers (as T/M ratios), we verified that the T/M ratio is highly correlated with absolute telomere length from the southern blot analysis. Together, the mcs primers were able to represent the telomere lengths accurately in cancer DNA samples. These results would allow for analyses of telomeres within cancerous DNA and the development of new, less invasive diagnostic tools for cancer. Neoplasia Press 2018-03-21 /pmc/articles/PMC5915991/ /pubmed/29573637 http://dx.doi.org/10.1016/j.neo.2018.02.007 Text en © 2018 The Authors http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Original article
Dahlgren, Paige N.
Bishop, Kanokwan
Dey, Shatovisha
Herbert, Brittney-Shea
Tanaka, Hiromi
Development of a New Monochrome Multiplex qPCR Method for Relative Telomere Length Measurement in Cancer
title Development of a New Monochrome Multiplex qPCR Method for Relative Telomere Length Measurement in Cancer
title_full Development of a New Monochrome Multiplex qPCR Method for Relative Telomere Length Measurement in Cancer
title_fullStr Development of a New Monochrome Multiplex qPCR Method for Relative Telomere Length Measurement in Cancer
title_full_unstemmed Development of a New Monochrome Multiplex qPCR Method for Relative Telomere Length Measurement in Cancer
title_short Development of a New Monochrome Multiplex qPCR Method for Relative Telomere Length Measurement in Cancer
title_sort development of a new monochrome multiplex qpcr method for relative telomere length measurement in cancer
topic Original article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915991/
https://www.ncbi.nlm.nih.gov/pubmed/29573637
http://dx.doi.org/10.1016/j.neo.2018.02.007
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