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

DeLorenzo, Drew M.; Moon, Tae Seok

doi:10.1038/s41598-018-24486-w

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

Journal Article · 16 April 2018 · Scientific Reports

DOI: https://doi.org/10.1038/s41598-018-24486-w · OSTI ID:1500023

DeLorenzo, Drew M. ^[1];

^[1]

Washington Univ., St. Louis, MO (United States)

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.

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Research Organization:: Washington Univ., St. Louis, MO (United States)

Sponsoring Organization:: USDOE

Grant/Contract Number:: SC0012705; SC0018324

OSTI ID:: 1500023

Journal Information:: Scientific Reports, Vol. 8, Issue 1; ISSN 2045-2322

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 16 works

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