Selection and Evaluation of Reference Genes for Reverse Transcription-Quantitative PCR Expression Studies in a Thermophilic Bacterium Grown under Different Culture Conditions.
PLoS One
; 10(6): e0131015, 2015.
Article
em En
| MEDLINE
| ID: mdl-26115538
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Regulação Bacteriana da Expressão Gênica
/
Reação em Cadeia da Polimerase Via Transcriptase Reversa
/
Perfilação da Expressão Gênica
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Deinococcus
Idioma:
En
Revista:
PLoS One
Assunto da revista:
CIENCIA
/
MEDICINA
Ano de publicação:
2015
Tipo de documento:
Article
País de afiliação:
Estados Unidos