Selection and validation of reference genes suitable for gene expression analysis by Reverse Transcription Quantitative real-time PCR in Acinetobacter baumannii
Abstract 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 Tran...
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| Format: | Article |
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Nature Portfolio
2024-02-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-024-51499-5 |
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| author | Paloma Aparecida Alves de Oliveira Juliana Baboghlian Clarissa Orandina Aparecida Ramos Alquiandra Stefani Ferreira Mançano Andréia de Melo Porcari Raquel Girardello Lúcio Fábio Caldas Ferraz |
| author_facet | Paloma Aparecida Alves de Oliveira Juliana Baboghlian Clarissa Orandina Aparecida Ramos Alquiandra Stefani Ferreira Mançano Andréia de Melo Porcari Raquel Girardello Lúcio Fábio Caldas Ferraz |
| author_sort | Paloma Aparecida Alves de Oliveira |
| collection | DOAJ |
| description | Abstract 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 CT, 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. |
| first_indexed | 2024-03-07T15:04:59Z |
| format | Article |
| id | doaj.art-18e5a2aa5ef1425ebc0354f87bc5b411 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-07T15:04:59Z |
| publishDate | 2024-02-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-18e5a2aa5ef1425ebc0354f87bc5b4112024-03-05T18:56:00ZengNature PortfolioScientific Reports2045-23222024-02-0114111110.1038/s41598-024-51499-5Selection and validation of reference genes suitable for gene expression analysis by Reverse Transcription Quantitative real-time PCR in Acinetobacter baumanniiPaloma Aparecida Alves de Oliveira0Juliana Baboghlian1Clarissa Orandina Aparecida Ramos2Alquiandra Stefani Ferreira Mançano3Andréia de Melo Porcari4Raquel Girardello5Lúcio Fábio Caldas Ferraz6Laboratório de Biologia Molecular de Microrganismos, Universidade São FranciscoLaboratório de Biologia Molecular de Microrganismos, Universidade São FranciscoLaboratório de Biologia Molecular de Microrganismos, Universidade São FranciscoLaboratório de Biologia Molecular de Microrganismos, Universidade São FranciscoLaboratório Multidisciplinar de Pesquisa, Universidade São FranciscoLaboratório de Biologia Molecular de Microrganismos, Universidade São FranciscoLaboratório de Biologia Molecular de Microrganismos, Universidade São FranciscoAbstract 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 CT, 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.https://doi.org/10.1038/s41598-024-51499-5 |
| spellingShingle | Paloma Aparecida Alves de Oliveira Juliana Baboghlian Clarissa Orandina Aparecida Ramos Alquiandra Stefani Ferreira Mançano Andréia de Melo Porcari Raquel Girardello Lúcio Fábio Caldas Ferraz Selection and validation of reference genes suitable for gene expression analysis by Reverse Transcription Quantitative real-time PCR in Acinetobacter baumannii Scientific Reports |
| title | Selection and validation of reference genes suitable for gene expression analysis by Reverse Transcription Quantitative real-time PCR in Acinetobacter baumannii |
| title_full | Selection and validation of reference genes suitable for gene expression analysis by Reverse Transcription Quantitative real-time PCR in Acinetobacter baumannii |
| title_fullStr | Selection and validation of reference genes suitable for gene expression analysis by Reverse Transcription Quantitative real-time PCR in Acinetobacter baumannii |
| title_full_unstemmed | Selection and validation of reference genes suitable for gene expression analysis by Reverse Transcription Quantitative real-time PCR in Acinetobacter baumannii |
| title_short | Selection and validation of reference genes suitable for gene expression analysis by Reverse Transcription Quantitative real-time PCR in Acinetobacter baumannii |
| title_sort | selection and validation of reference genes suitable for gene expression analysis by reverse transcription quantitative real time pcr in acinetobacter baumannii |
| url | https://doi.org/10.1038/s41598-024-51499-5 |
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