Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630

Abstract Rhodococcus opacus PD630 is a gram-positive bacterium with promising attributes for the conversion of lignin into valuable fuels and chemicals. To develop an organism as a cellular factory, it is necessary to have a deep understanding of its metabolism and any heterologous pathways being ex...

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Autores principales:	Drew M. DeLorenzo, Tae Seok Moon
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/dca3f2c5a0464dbb8f2923d9df622fc9
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spelling	oai:doaj.org-article:dca3f2c5a0464dbb8f2923d9df622fc92021-12-02T15:08:14ZSelection of stable reference genes for RT-qPCR in Rhodococcus opacus PD63010.1038/s41598-018-24486-w2045-2322https://doaj.org/article/dca3f2c5a0464dbb8f2923d9df622fc92018-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-24486-whttps://doaj.org/toc/2045-2322Abstract Rhodococcus opacus PD630 is a gram-positive bacterium with promising attributes for the conversion of lignin into valuable fuels and chemicals. To develop an organism as a cellular factory, it is necessary to have a deep understanding of its metabolism and any heterologous pathways being expressed. For the purpose of quantifying gene transcription, reverse transcription quantitative PCR (RT-qPCR) is the gold standard due to its sensitivity and reproducibility. However, RT-qPCR requires the use of reference genes whose expression is stable across distinct growth or treatment conditions to normalize the results. Unfortunately, no in-depth analysis of stable reference genes has been conducted in Rhodococcus, inhibiting the utilization of RT-qPCR in R. opacus. In this work, ten candidate reference genes, chosen based on previously collected RNA sequencing data or literature, were examined under four distinct growth conditions using three mathematical programs (BestKeeper, Normfinder, and geNorm). Based on this analysis, the minimum number of reference genes required was found to be two, and two separate pairs of references genes were identified as optimal normalization factors for when ribosomal RNA is either present or depleted. This work represents the first validation of reference genes for Rhodococcus, providing a valuable starting point for future research.Drew M. DeLorenzoTae Seok MoonNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-9 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Drew M. DeLorenzo Tae Seok Moon Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630
description	Abstract Rhodococcus opacus PD630 is a gram-positive bacterium with promising attributes for the conversion of lignin into valuable fuels and chemicals. To develop an organism as a cellular factory, it is necessary to have a deep understanding of its metabolism and any heterologous pathways being expressed. For the purpose of quantifying gene transcription, reverse transcription quantitative PCR (RT-qPCR) is the gold standard due to its sensitivity and reproducibility. However, RT-qPCR requires the use of reference genes whose expression is stable across distinct growth or treatment conditions to normalize the results. Unfortunately, no in-depth analysis of stable reference genes has been conducted in Rhodococcus, inhibiting the utilization of RT-qPCR in R. opacus. In this work, ten candidate reference genes, chosen based on previously collected RNA sequencing data or literature, were examined under four distinct growth conditions using three mathematical programs (BestKeeper, Normfinder, and geNorm). Based on this analysis, the minimum number of reference genes required was found to be two, and two separate pairs of references genes were identified as optimal normalization factors for when ribosomal RNA is either present or depleted. This work represents the first validation of reference genes for Rhodococcus, providing a valuable starting point for future research.
format	article
author	Drew M. DeLorenzo Tae Seok Moon
author_facet	Drew M. DeLorenzo Tae Seok Moon
author_sort	Drew M. DeLorenzo
title	Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630
title_short	Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630
title_full	Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630
title_fullStr	Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630
title_full_unstemmed	Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630
title_sort	selection of stable reference genes for rt-qpcr in rhodococcus opacus pd630
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/dca3f2c5a0464dbb8f2923d9df622fc9
work_keys_str_mv	AT drewmdelorenzo selectionofstablereferencegenesforrtqpcrinrhodococcusopacuspd630 AT taeseokmoon selectionofstablereferencegenesforrtqpcrinrhodococcusopacuspd630
_version_	1718388219359264768

Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630

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