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Title: 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 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.

Authors:
 [1]; ORCiD logo [1]
  1. Washington Univ., St. Louis, MO (United States)
Publication Date:
Research Org.:
Washington Univ., St. Louis, MO (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1500023
Grant/Contract Number:  
SC0012705; SC0018324
Resource Type:
Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 8; Journal Issue: 1; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

DeLorenzo, Drew M., and Moon, Tae Seok. Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630. United States: N. p., 2018. Web. doi:10.1038/s41598-018-24486-w.
DeLorenzo, Drew M., & Moon, Tae Seok. Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630. United States. doi:10.1038/s41598-018-24486-w.
DeLorenzo, Drew M., and Moon, Tae Seok. Mon . "Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630". United States. doi:10.1038/s41598-018-24486-w. https://www.osti.gov/servlets/purl/1500023.
@article{osti_1500023,
title = {Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630},
author = {DeLorenzo, Drew M. and Moon, Tae Seok},
abstractNote = {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.},
doi = {10.1038/s41598-018-24486-w},
journal = {Scientific Reports},
number = 1,
volume = 8,
place = {United States},
year = {2018},
month = {4}
}

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Works referenced in this record:

Real time quantitative PCR.
journal, October 1996

  • Heid, C. A.; Stevens, J.; Livak, K. J.
  • Genome Research, Vol. 6, Issue 10, p. 986-994
  • DOI: 10.1101/gr.6.10.986