Selection and validation of reference genes for gene expression studies in Klebsiella pneumoniae using Reverse Transcription Quantitative real-time PCR
| Autor: | Leonardo Prado Stuchi, Marcelo Lima Ribeiro, Lúcio Fábio Caldas Ferraz, Ana Érika Inácio Gomes, Renato Vicentini, João Batista Henrique, Nathália Maria Gonçalves Siqueira, Michelle Darrieux |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Candidate gene Klebsiella pneumoniae 030106 microbiology lcsh:Medicine Computational biology Real-Time Polymerase Chain Reaction law.invention Database normalization 03 medical and health sciences law Reference genes Gene expression lcsh:Science Gene Polymerase chain reaction Regulation of gene expression Multidisciplinary biology Reverse Transcriptase Polymerase Chain Reaction Gene Expression Profiling lcsh:R Computational Biology Reproducibility of Results Gene Expression Regulation Bacterial Reference Standards biology.organism_classification Genes Bacterial lcsh:Q Algorithms |
| Zdroj: | Scientific Reports, Vol 8, Iss 1, Pp 1-14 (2018) |
| ISSN: | 2045-2322 |
| Popis: | 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 CT 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. |
| Databáze: | OpenAIRE |
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