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Case Study Using Recommended Reference Genes Actin and 18S for Reverse-Transcription Quantitative Real-Time PCR Analysis in Myzus persicae

Myzus persicae is a globally important pest with the ability to adjust to a wide range of environmental situations, and many molecular technologies have been developed and applied to understand the biology and/or control this pest insect directly. Reverse-transcription quantitative real-time PCR (RT...

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Autores principales: Rahman, Saqib, Zhao, Zhenzhen, Umair Sial, Muhammad, Zhang, Yanning, Jiang, Hongyun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8528319/
https://www.ncbi.nlm.nih.gov/pubmed/34669698
http://dx.doi.org/10.1371/journal.pone.0258201
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author Rahman, Saqib
Zhao, Zhenzhen
Umair Sial, Muhammad
Zhang, Yanning
Jiang, Hongyun
author_facet Rahman, Saqib
Zhao, Zhenzhen
Umair Sial, Muhammad
Zhang, Yanning
Jiang, Hongyun
author_sort Rahman, Saqib
collection PubMed
description Myzus persicae is a globally important pest with the ability to adjust to a wide range of environmental situations, and many molecular technologies have been developed and applied to understand the biology and/or control this pest insect directly. Reverse-transcription quantitative real-time PCR (RT-qPCR) is a primary molecular technology that is used to quantify gene expression. Choosing a stable reference gene is significantly important for precisely clarifying the expression level of the target gene. Actin and 18S have been recommended as stable compounds for real-time RT-qPCR in M. persicae under the tested biotic and abiotic conditions. In this study, we checked the stability of Actin and 18S by analyzing the relative expression levels of the cytochrome 450 monooxygenase family member genes CYP6CY3 and CYP6-1, carboxylesterase gene E4 and vacuolar protein sorting gene VPS11 via RT-qPCR under various conditions. The expression levels of these four target genes were normalized using both Actin and 18S individually and the combination of these two genes. Our results confirmed that Actin and 18S can be used as reference genes to normalize the expression of target genes under insecticide treatment and starvation in M. persicae. However, at the developmental stages of M. persicae, the expression of the four tested target genes was normalized stably by Actin but not 18S, with the latter presenting a problematic change with the developmental stages. Thus, the stability of reference genes in response to diverse biotic and abiotic factors should be evaluated before each RT-qPCR experiment.
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spelling pubmed-85283192021-10-21 Case Study Using Recommended Reference Genes Actin and 18S for Reverse-Transcription Quantitative Real-Time PCR Analysis in Myzus persicae Rahman, Saqib Zhao, Zhenzhen Umair Sial, Muhammad Zhang, Yanning Jiang, Hongyun PLoS One Research Article Myzus persicae is a globally important pest with the ability to adjust to a wide range of environmental situations, and many molecular technologies have been developed and applied to understand the biology and/or control this pest insect directly. Reverse-transcription quantitative real-time PCR (RT-qPCR) is a primary molecular technology that is used to quantify gene expression. Choosing a stable reference gene is significantly important for precisely clarifying the expression level of the target gene. Actin and 18S have been recommended as stable compounds for real-time RT-qPCR in M. persicae under the tested biotic and abiotic conditions. In this study, we checked the stability of Actin and 18S by analyzing the relative expression levels of the cytochrome 450 monooxygenase family member genes CYP6CY3 and CYP6-1, carboxylesterase gene E4 and vacuolar protein sorting gene VPS11 via RT-qPCR under various conditions. The expression levels of these four target genes were normalized using both Actin and 18S individually and the combination of these two genes. Our results confirmed that Actin and 18S can be used as reference genes to normalize the expression of target genes under insecticide treatment and starvation in M. persicae. However, at the developmental stages of M. persicae, the expression of the four tested target genes was normalized stably by Actin but not 18S, with the latter presenting a problematic change with the developmental stages. Thus, the stability of reference genes in response to diverse biotic and abiotic factors should be evaluated before each RT-qPCR experiment. Public Library of Science 2021-10-20 /pmc/articles/PMC8528319/ /pubmed/34669698 http://dx.doi.org/10.1371/journal.pone.0258201 Text en © 2021 Rahman et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Rahman, Saqib
Zhao, Zhenzhen
Umair Sial, Muhammad
Zhang, Yanning
Jiang, Hongyun
Case Study Using Recommended Reference Genes Actin and 18S for Reverse-Transcription Quantitative Real-Time PCR Analysis in Myzus persicae
title Case Study Using Recommended Reference Genes Actin and 18S for Reverse-Transcription Quantitative Real-Time PCR Analysis in Myzus persicae
title_full Case Study Using Recommended Reference Genes Actin and 18S for Reverse-Transcription Quantitative Real-Time PCR Analysis in Myzus persicae
title_fullStr Case Study Using Recommended Reference Genes Actin and 18S for Reverse-Transcription Quantitative Real-Time PCR Analysis in Myzus persicae
title_full_unstemmed Case Study Using Recommended Reference Genes Actin and 18S for Reverse-Transcription Quantitative Real-Time PCR Analysis in Myzus persicae
title_short Case Study Using Recommended Reference Genes Actin and 18S for Reverse-Transcription Quantitative Real-Time PCR Analysis in Myzus persicae
title_sort case study using recommended reference genes actin and 18s for reverse-transcription quantitative real-time pcr analysis in myzus persicae
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8528319/
https://www.ncbi.nlm.nih.gov/pubmed/34669698
http://dx.doi.org/10.1371/journal.pone.0258201
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