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.

Figure 1

Confirmation of specificity of primers used in RT-qPCR analysis. (A) Contrast enhanced image of electrophoresis gel confirming amplicon size and primer specificity using RT-qPCR amplification product. The sizes for nucleotide ladder are indicated to left of bands (50 to 200 bp). The size of each amplicon is denoted in Table 1. The original non-enhanced gel image, in addition to the corresponding gel image of no template controls (NTCs), can be found in Supplementary Figure 5. (B) Representative melt curves post RT-qPCR for RG1 and RG2 as well as the corresponding NTCs (see Supplementary Figure 2 for all melt curves).

Figure 2

C_T values for ten candidate reference genes in R. opacus. RNA was extracted from biological triplicates of R. opacus grown in four distinct growth conditions (HN [circle], LN [triangle], TSB [diamond], and PHE [square]; see Materials and Methods for full description). RT-qPCR was performed in technical triplicate on each biological replicate. Each point represents the average threshold cycle (C_T) value of all replicates for the listed gene and growth condition. Error bars represent one standard deviation.

Figure 3

Rankings of candidate reference genes. Genes were ranked from the least stable (on the left) to the most stable (on the right). Analysis was performed after pooling C_T data across all four growth conditions. The designation of 1 means that the analyses were performed on all ten candidate RGs. The designation of 2 means that the analyses were performed on the eight non-rRNA candidate RGs. (A) Genes were ranked according to their BestKeeper r-value. BestKeeper r-value significance can be found in Supplementary Tables 3 and 4. (B) Genes were ranked according to their Normfinder stability value. (C) Genes were ranked according to their geNorm M value.

Figure 4

Minimum number of reference genes. The pair-wise variation Vn/Vn + 1, where n represents the number of RGs used in the normalization factor (NF), was calculated by geNorm to determine the minimum number of RGs required for normalization. A geNorm V value below 0.15 signifies that no additional benefit is gained from increasing the number of reference genes from n to n + 1. The dashed line represents a V value of 0.15.

Figure 5

Effect of reference gene choice on RT-qPCR normalization. (A) Box plots of averaged PD630_RS15810 expression data (C_T) for all four growth conditions (HN, LN, TSB, and PHE). Each gray box represents the first through third quartiles, the solid black line represents the median, and the whiskers represent the minimum and maximum values. (B–D) The normalized relative expression ratio of PD630_LPD05540 going from HN to either LN (B), TSB (C), or PHE (D). The expression data was normalized with a NF, including RG6, RG10, RG3/RG7, RG3/RG8, RG7/RG8, and RG3/RG7/RG8 using REST 2009. Error bars represent the 95% confidence interval (CI) as calculated by REST 2009. Stars indicate that a 95% CI range falls outside of the 95% CI range of the RG3/RG7/RG8 (and RG7/RG8) normalized ratio.