Selection and validation of reference genes for gene expression studies in Klebsiella pneumoniae using Reverse Transcription Quantitative real-time PCR

• Published in: Scientific reports Vol. 8; no. 1; pp. 9001 - 14
• Main Authors: Gomes, Ana Érika Inácio, Stuchi, Leonardo Prado, Siqueira, Nathália Maria Gonçalves, Henrique, João Batista, Vicentini, Renato, Ribeiro, Marcelo Lima, Darrieux, Michelle, Ferraz, Lúcio Fábio Caldas
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 13-06-2018; Nature Publishing Group
• Subjects: 38/39; 38/90; 45/29; 45/44; 631/1647/2017/2003; 631/208/199; 631/208/325/1506; 631/337/572/2102; 82/80; 82/83; Gene expression; Humanities and Social Sciences; Iron; Klebsiella pneumoniae; multidisciplinary; Polymerase chain reaction; RecA protein; Reverse transcription; Science; Science (multidisciplinary); Statistical analysis; Statistics
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-018-27420-2

For reliable results, Reverse Transcription Quantitative real-time Polymerase Chain Reaction (RT-qPCR) analyses depend on stably expressed reference genes for data normalization purposes.
Klebsiella pneumoniae
is an opportunistic Gram-negative bacterium that has become a serious threat worldwide. Unfortunately, there is no consensus for an ideal reference gene for RT-qPCR data normalization on
K. pneumoniae
. In this study, the expression profile of eleven candidate reference genes was assessed in
K. pneumoniae
cells submitted to various experimental conditions, and the expression stability of these candidate genes was evaluated using statistical algorithms BestKeeper, NormFinder, geNorm, Delta C
T
and RefFinder. The statistical analyses ranked
recA
,
rho
,
proC
and
rpoD
as the most suitable reference genes for accurate RT-qPCR data normalization in
K. pneumoniae
. The reliability of the proposed reference genes was validated by normalizing the relative expression of iron-regulated genes in
K. pneumoniae
cells submitted to iron-replete and iron-limited conditions. This work emphasizes that the stable expression of any potential reference candidate gene must be validated in each physiological condition or experimental treatment under study.