Cargando…

Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR

Reverse transcription quantitative PCR (RT-qPCR), a method of choice for quantification of gene expression changes, requires stably expressed reference genes for normalization of data. So far, no reference genes were established for the Alphaproteobacteria of the genus Ochrobactrum. Here, we determi...

Descripción completa

Detalles Bibliográficos
Autores principales: Krzyżanowska, Dorota M., Supernat, Anna, Maciąg, Tomasz, Matuszewska, Marta, Jafra, Sylwia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739375/
https://www.ncbi.nlm.nih.gov/pubmed/31511547
http://dx.doi.org/10.1038/s41598-019-49474-6
_version_ 1783450931221757952
author Krzyżanowska, Dorota M.
Supernat, Anna
Maciąg, Tomasz
Matuszewska, Marta
Jafra, Sylwia
author_facet Krzyżanowska, Dorota M.
Supernat, Anna
Maciąg, Tomasz
Matuszewska, Marta
Jafra, Sylwia
author_sort Krzyżanowska, Dorota M.
collection PubMed
description Reverse transcription quantitative PCR (RT-qPCR), a method of choice for quantification of gene expression changes, requires stably expressed reference genes for normalization of data. So far, no reference genes were established for the Alphaproteobacteria of the genus Ochrobactrum. Here, we determined reference genes for gene expression studies in O. quorumnocens A44. Strain A44 was cultured under 10 different conditions and the stability of expression of 11 candidate genes was evaluated using geNorm, NormFinder and BestKeeper. Most stably expressed genes were found to be rho, gyrB and rpoD. Our results can facilitate the choice of reference genes in the related Ochrobactrum strains. O. quorumnocens A44 is able to inactivate a broad spectrum of N-acyl homoserine lactones (AHLs) – the quorum sensing molecules of many Gram-negative bacteria. This activity is attributed to AiiO hydrolase, yet it remains unclear whether AHLs are the primary substrate of this enzyme. Using the established RT-qPCR setup, we found that the expression of the aiiO gene upon exposure to two AHLs, C6-HLS and 3OC12-HSL, does not change above the 1-fold significance threshold. The implications of this finding are discussed in the light of the role of quorum sensing-interfering enzymes in the host strains.
format Online
Article
Text
id pubmed-6739375
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher Nature Publishing Group UK
record_format MEDLINE/PubMed
spelling pubmed-67393752019-09-22 Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR Krzyżanowska, Dorota M. Supernat, Anna Maciąg, Tomasz Matuszewska, Marta Jafra, Sylwia Sci Rep Article Reverse transcription quantitative PCR (RT-qPCR), a method of choice for quantification of gene expression changes, requires stably expressed reference genes for normalization of data. So far, no reference genes were established for the Alphaproteobacteria of the genus Ochrobactrum. Here, we determined reference genes for gene expression studies in O. quorumnocens A44. Strain A44 was cultured under 10 different conditions and the stability of expression of 11 candidate genes was evaluated using geNorm, NormFinder and BestKeeper. Most stably expressed genes were found to be rho, gyrB and rpoD. Our results can facilitate the choice of reference genes in the related Ochrobactrum strains. O. quorumnocens A44 is able to inactivate a broad spectrum of N-acyl homoserine lactones (AHLs) – the quorum sensing molecules of many Gram-negative bacteria. This activity is attributed to AiiO hydrolase, yet it remains unclear whether AHLs are the primary substrate of this enzyme. Using the established RT-qPCR setup, we found that the expression of the aiiO gene upon exposure to two AHLs, C6-HLS and 3OC12-HSL, does not change above the 1-fold significance threshold. The implications of this finding are discussed in the light of the role of quorum sensing-interfering enzymes in the host strains. Nature Publishing Group UK 2019-09-11 /pmc/articles/PMC6739375/ /pubmed/31511547 http://dx.doi.org/10.1038/s41598-019-49474-6 Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Krzyżanowska, Dorota M.
Supernat, Anna
Maciąg, Tomasz
Matuszewska, Marta
Jafra, Sylwia
Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR
title Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR
title_full Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR
title_fullStr Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR
title_full_unstemmed Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR
title_short Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR
title_sort selection of reference genes for measuring the expression of aiio in ochrobactrum quorumnocens a44 using rt-qpcr
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739375/
https://www.ncbi.nlm.nih.gov/pubmed/31511547
http://dx.doi.org/10.1038/s41598-019-49474-6
work_keys_str_mv AT krzyzanowskadorotam selectionofreferencegenesformeasuringtheexpressionofaiioinochrobactrumquorumnocensa44usingrtqpcr
AT supernatanna selectionofreferencegenesformeasuringtheexpressionofaiioinochrobactrumquorumnocensa44usingrtqpcr
AT maciagtomasz selectionofreferencegenesformeasuringtheexpressionofaiioinochrobactrumquorumnocensa44usingrtqpcr
AT matuszewskamarta selectionofreferencegenesformeasuringtheexpressionofaiioinochrobactrumquorumnocensa44usingrtqpcr
AT jafrasylwia selectionofreferencegenesformeasuringtheexpressionofaiioinochrobactrumquorumnocensa44usingrtqpcr