Selection and validation of reference genes suitable for gene expression analysis by Reverse Transcription Quantitative real-time PCR in Acinetobacter baumannii

• Published in: Scientific reports Vol. 14; no. 1; p. 3830
• Main Authors: de Oliveira, Paloma Aparecida Alves, Baboghlian, Juliana, Ramos, Clarissa Orandina Aparecida, Mançano, Alquiandra Stefani Ferreira, Porcari, Andréia de Melo, Girardello, Raquel, Ferraz, Lúcio Fábio Caldas
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 15.02.2024; Nature Portfolio
• Subjects: Acinetobacter baumannii; Acinetobacter baumannii - genetics; Algorithms; Drug resistance; Environmental conditions; Gene expression; Gene Expression Profiling; Gram-negative bacteria; Membrane proteins; Multidrug resistance; Nosocomial infection; OmpA protein; Real-Time Polymerase Chain Reaction; Reference Standards; Reverse Transcription; RpoB protein; Statistical analysis; Statistics
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-024-51499-5

Acinetobacter baumannii is a Gram-negative bacterium considered an emerging multi-drug-resistant pathogen. Furthermore, this bacterium can survive in extreme environmental conditions, which makes it a frequent cause of nosocomial infection outbreaks. Gene expression analyses by Reverse Transcription Quantitative real-time PCR (RT-qPCR) depend on a reference gene, also called an endogenous gene, which is used to normalize the generated data and thus ensure an accurate analysis with minimal errors. Currently, gene expression analyses in A. baumannii are compromised, as there are no reports in the literature describing the identification of validated reference genes for use in RT-qPCR analyses. For this reason, we selected twelve candidate reference genes of A. baumannii and assessed their expression profile under different experimental and culture conditions. The expression stability of the candidate genes was evaluated by using statistical algorithms such as BestKeeper, geNorm, NormFinder, Delta C , and RefFinder, in order to identify the most suitable candidate reference genes for RT-qPCR analyses. The statistical analyses indicated rpoB, rpoD, and fabD genes as the most adequate to ensure accurate normalization of RT-qPCR data in A. baumannii. The accuracy of the proposed reference genes was validated by using them to normalize the expression of the ompA gene, encoding the outer membrane protein A, in A. baumannii sensible and resistant to the antibiotic polymyxin. The present work provides suitable reference genes for precise RT-qPCR data normalization on future gene expression studies with A. baumannii.