Selection and Evaluation of Reference Genes for Reverse Transcription-Quantitative PCR Expression Studies in a Thermophilic Bacterium Grown under Different Culture Conditions

Abstract

The phylum Deinococcus-Thermus is a deeply-branching lineage of bacteria widely recognized as one of the most extremophilic. Members of the Thermus genus are of major interest due to both their bioremediation and biotechnology potentials. However, the molecular mechanisms associated with these key metabolic pathways remain unknown. Reverse-transcription quantitative PCR (RT-qPCR) is a high-throughput means of studying the expression of a large suite of genes over time and under different conditions. The selection of a stably-expressed reference gene is critical when using relative quantification methods, as target gene expression is normalized to expression of the reference gene. However, little information exists as to reference gene selection in extremophiles. This study evaluated 11 candidate reference genes for use with the thermophile Thermus scotoductus when grown under different culture conditions. Based on the combined stability values from BestKeeper and NormFinder software packages, the following are the most appropriate reference genes when comparing: (1) aerobic and anaerobic growth: TSC_c19900, polA2, gyrA, gyrB; (2) anaerobic growth with varied electron acceptors: TSC_c19900, infA, pfk, gyrA, gyrB; (3) aerobic growth with different heating methods: gyrA, gap, gyrB; (4) all conditions mentioned above: gap, gyrA, gyrB. The commonly-employed rpoC does not serve as a reliable reference gene in thermophiles, due to its expression instability across all culture conditions tested here. As extremophiles exhibit a tendency for polyploidy, absolute quantification was employed to determine the ratio of transcript to gene copy number in a subset of the genes. A strong negative correlation was found to exist between ratio and threshold cycle (CT) values, demonstrating that CT changes reflect transcript copy number, and not gene copy number, fluctuations. Even with the potential for polyploidy in extremophiles, the results obtained via absolute quantification indicate that relative quantification is appropriate for RT-qPCR studies with this thermophile.

Dual energy metabolism of the Campylobacterota endosymbiont in the chemosynthetic snail Alviniconcha marisindica

“…In addition, to normalize the comparative expression abundance of the functional genes between both kAlv and eAlv endosymbionts, we used gyrB and gap genes, which encode the DNA gyrase B subunit and glyceraldehyde 3phosphate dehydrogenase, respectively. These genes are widely used as internal controls for the functional gene expression of bacteria and humans [55][56][57][58]. The amplified fragments of gyrB (695 bp) and gap (620 bp) shared 99.6% (three-bases mismatch) and 99.8% (one-base mismatch), respectively, with the endosymbionts.…”

Low-Molecular-Weight Thiols and Thioredoxins Are Important Players in Hg(II) Resistance in Thermus thermophilus HB27

“…mRNA transcripts in WT cells exposed to 0 or 1 M Hg(II) for 7.5, 15, or 60 min were quantified, and transcript abundances were normalized to that of gyrA (see Table S1 in the supplemental material) (25). Mercury treatment increased transcript levels of all thiol-related genes tested, from over 10-fold for oah1 and trxA1 to about 2-fold for bshC (Fig.…”

Molecular Mechanisms Contributing to the Growth and Physiology of an Extremophile Cultured with Dielectric Heating

“…Total RNA was extracted with the RNeasy minikit using a previously optimized protocol (15). Briefly, 180 l of lysozyme (15 mg/ml) resuspended in TE buffer (30 mM Tris-HCl, 1 mM EDTA [pH 8]) plus 20 l proteinase K (Qiagen) was added to each cell pellet, and then the samples were incubated with agitation (125 rpm) for 25 min at room temperature.…”

“…Quantitative reverse transcription-PCR (qRT-PCR) assays based on SYBR green chemistry (19) were developed for a subset of genes at each of the three time points in order to verify RNA-seq results. Total RNA was converted to cDNA using the high-capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA) using a previously optimized protocol (15). The reaction mixtures were incubated at 37°C for 60 min, followed by heating to 95°C for 5 min in a 2720 thermocycler (Applied Biosystems).…”

“…Gene-specific primers, along with previously validated reference gene primers (15), are listed in Table 1. For each assay, primers were designed that targeted a ca.…”