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

Drew M Delorenzo,Tae Seok Moon

doi:10.1038/s41598-018-24486-w

Drew M Delorenzo, Tae Seok Moon

Open Access

https://doi.org/10.1038/s41598-018-24486-w

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Journal: Scientific Reports	Publication Date: Apr 16, 2018
Citations: 20	License type: open-access

Affiliation: Washington University in St. Louis

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.

Highlights

Rhodococcus opacus PD630 is a gram-positive, oleaginous bacterium that possesses beneficial traits for the conversion of lignin into valuable fuels and chemicals[1,2,3,4]
We identified ten candidate reference genes (RGs) and examined the stability of their expression in R. opacus across four distinct growth conditions using three mathematical models (BestKeeper[23], NormFinder[24], and geNorm16,25)
The expression of ten candidate reference genes was examined across four distinct growth conditions (HN, low nitrogen (LN), tryptic soy broth medium (TSB), and phenol and high nitrogen (PHE)) using three mathematical models (BestKeeper, Normfinder, and geNorm) to identify and rank gene expression stability

Summary

Introduction

Rhodococcus opacus PD630 (hereafter R. opacus) is a gram-positive, oleaginous bacterium that possesses beneficial traits for the conversion of lignin into valuable fuels and chemicals[1,2,3,4]. RNA sequencing (RNA-Seq) is a newer technology that has become the default method for examining the entire transcriptome of an organism[12] It can add additional costs if only several genes are of interest, is limited when mRNA concentrations are low ( this is changing with the advent of single cell sequencing13), and generally still requires corroboration via additional quantitative methods[12]. One such complimentary method is reverse transcription quantitative PCR (RT-qPCR), which is considered the gold standard of mRNA quantification due to its high sensitivity, reproducibility, speed, ability to examine numerous samples simultaneously, and large dynamic range[14,15]. This work facilitates the utilization of RT-qPCR in R. opacus, in addition to providing a valuable preliminary set of reference genes for further future validation in other Rhodococcus spp

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