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Identification and Validation of Reference Genes for Expression Analysis in Nitrogen-Fixing Bacteria under Environmental Stress

Reference genes, also referred to as housekeeping genes (HKGs), play an important role in gene expression analysis by serving as an internal control. These HKGs are usually involved in basic cellular functions and their expression should remain at relatively constant levels. Quantitative reverse tra...

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Autores principales: Parks, Dylan, Peterson, Christian, Chang, Woo-Suk
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9505014/
https://www.ncbi.nlm.nih.gov/pubmed/36143415
http://dx.doi.org/10.3390/life12091379
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author Parks, Dylan
Peterson, Christian
Chang, Woo-Suk
author_facet Parks, Dylan
Peterson, Christian
Chang, Woo-Suk
author_sort Parks, Dylan
collection PubMed
description Reference genes, also referred to as housekeeping genes (HKGs), play an important role in gene expression analysis by serving as an internal control. These HKGs are usually involved in basic cellular functions and their expression should remain at relatively constant levels. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) has been used to measure gene expression. Since the normalization of gene expression data depends on baseline expression of HKGs, it is important to identify and verify true HKGs for the qRT-PCR analysis. The goal of this study is to identify and confirm HKGs in Bradyrhizobium diazoefficiens, a nitrogen fixing bacterium which forms a symbiotic relationship with soybean. By revealing such HKGs, the normalization of gene expression would be more robust, reliable, and consistent. Here, we analyzed previous gene expression data for B. diazoefficiens under multiple environmental conditions. As a result, we identified seven constitutively expressed genes among 8453 genes across all conditions. Their fold-change values were within a range of −1.25-fold < x < 1.25-fold. We adopted GeNorm, NormFinder, and comparative ∆Ct methods to rank the seven candidate genes based on their expression stability. To validate these potential HKGs, we measured their expression in various experimental conditions, such as heat, pH, and heavy metal stress. The HKGs that were found in B. diazoefficiens were also applied in closely related species by identifying their homologs.
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spelling pubmed-95050142022-09-24 Identification and Validation of Reference Genes for Expression Analysis in Nitrogen-Fixing Bacteria under Environmental Stress Parks, Dylan Peterson, Christian Chang, Woo-Suk Life (Basel) Article Reference genes, also referred to as housekeeping genes (HKGs), play an important role in gene expression analysis by serving as an internal control. These HKGs are usually involved in basic cellular functions and their expression should remain at relatively constant levels. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) has been used to measure gene expression. Since the normalization of gene expression data depends on baseline expression of HKGs, it is important to identify and verify true HKGs for the qRT-PCR analysis. The goal of this study is to identify and confirm HKGs in Bradyrhizobium diazoefficiens, a nitrogen fixing bacterium which forms a symbiotic relationship with soybean. By revealing such HKGs, the normalization of gene expression would be more robust, reliable, and consistent. Here, we analyzed previous gene expression data for B. diazoefficiens under multiple environmental conditions. As a result, we identified seven constitutively expressed genes among 8453 genes across all conditions. Their fold-change values were within a range of −1.25-fold < x < 1.25-fold. We adopted GeNorm, NormFinder, and comparative ∆Ct methods to rank the seven candidate genes based on their expression stability. To validate these potential HKGs, we measured their expression in various experimental conditions, such as heat, pH, and heavy metal stress. The HKGs that were found in B. diazoefficiens were also applied in closely related species by identifying their homologs. MDPI 2022-09-05 /pmc/articles/PMC9505014/ /pubmed/36143415 http://dx.doi.org/10.3390/life12091379 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Parks, Dylan
Peterson, Christian
Chang, Woo-Suk
Identification and Validation of Reference Genes for Expression Analysis in Nitrogen-Fixing Bacteria under Environmental Stress
title Identification and Validation of Reference Genes for Expression Analysis in Nitrogen-Fixing Bacteria under Environmental Stress
title_full Identification and Validation of Reference Genes for Expression Analysis in Nitrogen-Fixing Bacteria under Environmental Stress
title_fullStr Identification and Validation of Reference Genes for Expression Analysis in Nitrogen-Fixing Bacteria under Environmental Stress
title_full_unstemmed Identification and Validation of Reference Genes for Expression Analysis in Nitrogen-Fixing Bacteria under Environmental Stress
title_short Identification and Validation of Reference Genes for Expression Analysis in Nitrogen-Fixing Bacteria under Environmental Stress
title_sort identification and validation of reference genes for expression analysis in nitrogen-fixing bacteria under environmental stress
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9505014/
https://www.ncbi.nlm.nih.gov/pubmed/36143415
http://dx.doi.org/10.3390/life12091379
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