Cargando…

A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors

An assay for telomerase activity based on asymmetric polymerase chain reaction (A-PCR) on magnetic beads (MBs) and subsequent application of cycling probe technology (CPT) is described. In this assay, the telomerase reaction products are immobilized on MBs, which are then washed to remove PCR inhibi...

Descripción completa

Detalles Bibliográficos
Autores principales: Yaku, Hidenobu, Murashima, Takashi, Miyoshi, Daisuke, Sugimoto, Naoki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6269933/
https://www.ncbi.nlm.nih.gov/pubmed/24071983
http://dx.doi.org/10.3390/molecules181011751
_version_ 1783376582436454400
author Yaku, Hidenobu
Murashima, Takashi
Miyoshi, Daisuke
Sugimoto, Naoki
author_facet Yaku, Hidenobu
Murashima, Takashi
Miyoshi, Daisuke
Sugimoto, Naoki
author_sort Yaku, Hidenobu
collection PubMed
description An assay for telomerase activity based on asymmetric polymerase chain reaction (A-PCR) on magnetic beads (MBs) and subsequent application of cycling probe technology (CPT) is described. In this assay, the telomerase reaction products are immobilized on MBs, which are then washed to remove PCR inhibitors that are commonly found in clinical samples. The guanine-rich sequences (5'-(TTAGGG)n-3') of the telomerase reaction products are then preferentially amplified by A-PCR, and the amplified products are subsequently detected via CPT, where a probe RNA with a fluorophore at the 5' end and a quencher at the 3' end is hydrolyzed by RNase H in the presence of the target DNA. The catalyst-mediated cleavage of the probe RNA enhances fluorescence from the 5' end of the probe. The assay allowed us to successfully detect HeLa cells selectively over normal human dermal fibroblast (NHDF) cells. Importantly, this selectivity produced identical results with regard to detection of HeLa cells in the absence and presence of excess NHDF cells; therefore, this assay can be used for practical clinical applications. The lower limit of detection for HeLa cells was 50 cells, which is lower than that achieved with a conventional telomeric repeat amplification protocol assay. Our assay also eliminated false-negative results caused by PCR inhibitors. Furthermore, we show that this assay is appropriate for screening among G-quadruplex ligands to find those that inhibit telomerase activity.
format Online
Article
Text
id pubmed-6269933
institution National Center for Biotechnology Information
language English
publishDate 2013
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-62699332018-12-18 A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors Yaku, Hidenobu Murashima, Takashi Miyoshi, Daisuke Sugimoto, Naoki Molecules Article An assay for telomerase activity based on asymmetric polymerase chain reaction (A-PCR) on magnetic beads (MBs) and subsequent application of cycling probe technology (CPT) is described. In this assay, the telomerase reaction products are immobilized on MBs, which are then washed to remove PCR inhibitors that are commonly found in clinical samples. The guanine-rich sequences (5'-(TTAGGG)n-3') of the telomerase reaction products are then preferentially amplified by A-PCR, and the amplified products are subsequently detected via CPT, where a probe RNA with a fluorophore at the 5' end and a quencher at the 3' end is hydrolyzed by RNase H in the presence of the target DNA. The catalyst-mediated cleavage of the probe RNA enhances fluorescence from the 5' end of the probe. The assay allowed us to successfully detect HeLa cells selectively over normal human dermal fibroblast (NHDF) cells. Importantly, this selectivity produced identical results with regard to detection of HeLa cells in the absence and presence of excess NHDF cells; therefore, this assay can be used for practical clinical applications. The lower limit of detection for HeLa cells was 50 cells, which is lower than that achieved with a conventional telomeric repeat amplification protocol assay. Our assay also eliminated false-negative results caused by PCR inhibitors. Furthermore, we show that this assay is appropriate for screening among G-quadruplex ligands to find those that inhibit telomerase activity. MDPI 2013-09-25 /pmc/articles/PMC6269933/ /pubmed/24071983 http://dx.doi.org/10.3390/molecules181011751 Text en © 2013 by the authors; licensee MDPI, Basel, Switzerland. http://creativecommons.org/licenses/by/3.0/ This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
spellingShingle Article
Yaku, Hidenobu
Murashima, Takashi
Miyoshi, Daisuke
Sugimoto, Naoki
A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors
title A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors
title_full A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors
title_fullStr A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors
title_full_unstemmed A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors
title_short A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors
title_sort highly sensitive telomerase activity assay that eliminates false-negative results caused by pcr inhibitors
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6269933/
https://www.ncbi.nlm.nih.gov/pubmed/24071983
http://dx.doi.org/10.3390/molecules181011751
work_keys_str_mv AT yakuhidenobu ahighlysensitivetelomeraseactivityassaythateliminatesfalsenegativeresultscausedbypcrinhibitors
AT murashimatakashi ahighlysensitivetelomeraseactivityassaythateliminatesfalsenegativeresultscausedbypcrinhibitors
AT miyoshidaisuke ahighlysensitivetelomeraseactivityassaythateliminatesfalsenegativeresultscausedbypcrinhibitors
AT sugimotonaoki ahighlysensitivetelomeraseactivityassaythateliminatesfalsenegativeresultscausedbypcrinhibitors
AT yakuhidenobu highlysensitivetelomeraseactivityassaythateliminatesfalsenegativeresultscausedbypcrinhibitors
AT murashimatakashi highlysensitivetelomeraseactivityassaythateliminatesfalsenegativeresultscausedbypcrinhibitors
AT miyoshidaisuke highlysensitivetelomeraseactivityassaythateliminatesfalsenegativeresultscausedbypcrinhibitors
AT sugimotonaoki highlysensitivetelomeraseactivityassaythateliminatesfalsenegativeresultscausedbypcrinhibitors