Real-time relative qPCR without reference to control samples and estimation of run-specific PCR parameters from run-internal mini-standard curves.

<h4>Background</h4>Real-Time quantitative PCR is an important tool in research and clinical settings. Here, we describe two new approaches that broaden the scope of real-time quantitative PCR; namely, run-internal mini standard curves (RIMS) and direct real-time relative quantitative PCR...

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Autores principales:	Jens Magnus Bernth Jensen, Mikkel Steen Petersen, Marc Stegger, Lars J Østergaard, Bjarne K Møller
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e91401e5d8564dbb95be9489ab8cde4f
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spelling	oai:doaj.org-article:e91401e5d8564dbb95be9489ab8cde4f2021-12-02T20:19:52ZReal-time relative qPCR without reference to control samples and estimation of run-specific PCR parameters from run-internal mini-standard curves.1932-620310.1371/journal.pone.0011723https://doaj.org/article/e91401e5d8564dbb95be9489ab8cde4f2010-07-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20661435/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Real-Time quantitative PCR is an important tool in research and clinical settings. Here, we describe two new approaches that broaden the scope of real-time quantitative PCR; namely, run-internal mini standard curves (RIMS) and direct real-time relative quantitative PCR (drqPCR). RIMS are an efficient alternative to traditional standard curves and provide both run-specific and target-specific estimates of PCR parameters. The drqPCR enables direct estimation of target ratios without reference to conventional control samples.<h4>Methodology/principal findings</h4>In this study, we compared RIMS-based drqPCR with classical quantifications based on external standard curves and the "comparative Ct method". Specifically, we used a raw real-time PCR dataset as the basis for more than two-and-a-half million simulated quantifications with various user-defined conditions. Compared with classical approaches, we found that RIMS-based drqPCR provided superior precision and comparable accuracy.<h4>Conclusions/significance</h4>The obviation of referencing to control samples is attractive whenever unpaired samples are quantified. This may be in clinical and research settings; for instance, studies on chimerism, TREC quantifications, copy number variations etc. Also, lab-to-lab comparability can be greatly simplified.Jens Magnus Bernth JensenMikkel Steen PetersenMarc SteggerLars J ØstergaardBjarne K MøllerPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 5, Iss 7, p e11723 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Jens Magnus Bernth Jensen Mikkel Steen Petersen Marc Stegger Lars J Østergaard Bjarne K Møller Real-time relative qPCR without reference to control samples and estimation of run-specific PCR parameters from run-internal mini-standard curves.
description	<h4>Background</h4>Real-Time quantitative PCR is an important tool in research and clinical settings. Here, we describe two new approaches that broaden the scope of real-time quantitative PCR; namely, run-internal mini standard curves (RIMS) and direct real-time relative quantitative PCR (drqPCR). RIMS are an efficient alternative to traditional standard curves and provide both run-specific and target-specific estimates of PCR parameters. The drqPCR enables direct estimation of target ratios without reference to conventional control samples.<h4>Methodology/principal findings</h4>In this study, we compared RIMS-based drqPCR with classical quantifications based on external standard curves and the "comparative Ct method". Specifically, we used a raw real-time PCR dataset as the basis for more than two-and-a-half million simulated quantifications with various user-defined conditions. Compared with classical approaches, we found that RIMS-based drqPCR provided superior precision and comparable accuracy.<h4>Conclusions/significance</h4>The obviation of referencing to control samples is attractive whenever unpaired samples are quantified. This may be in clinical and research settings; for instance, studies on chimerism, TREC quantifications, copy number variations etc. Also, lab-to-lab comparability can be greatly simplified.
format	article
author	Jens Magnus Bernth Jensen Mikkel Steen Petersen Marc Stegger Lars J Østergaard Bjarne K Møller
author_facet	Jens Magnus Bernth Jensen Mikkel Steen Petersen Marc Stegger Lars J Østergaard Bjarne K Møller
author_sort	Jens Magnus Bernth Jensen
title	Real-time relative qPCR without reference to control samples and estimation of run-specific PCR parameters from run-internal mini-standard curves.
title_short	Real-time relative qPCR without reference to control samples and estimation of run-specific PCR parameters from run-internal mini-standard curves.
title_full	Real-time relative qPCR without reference to control samples and estimation of run-specific PCR parameters from run-internal mini-standard curves.
title_fullStr	Real-time relative qPCR without reference to control samples and estimation of run-specific PCR parameters from run-internal mini-standard curves.
title_full_unstemmed	Real-time relative qPCR without reference to control samples and estimation of run-specific PCR parameters from run-internal mini-standard curves.
title_sort	real-time relative qpcr without reference to control samples and estimation of run-specific pcr parameters from run-internal mini-standard curves.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/e91401e5d8564dbb95be9489ab8cde4f
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Real-time relative qPCR without reference to control samples and estimation of run-specific PCR parameters from run-internal mini-standard curves.

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