Cargando…
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...
Autores principales: | , , , , |
---|---|
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 |
_version_ | 1784586228292648960 |
---|---|
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. |
format | Online Article Text |
id | pubmed-8528319 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT rahmansaqib casestudyusingrecommendedreferencegenesactinand18sforreversetranscriptionquantitativerealtimepcranalysisinmyzuspersicae AT zhaozhenzhen casestudyusingrecommendedreferencegenesactinand18sforreversetranscriptionquantitativerealtimepcranalysisinmyzuspersicae AT umairsialmuhammad casestudyusingrecommendedreferencegenesactinand18sforreversetranscriptionquantitativerealtimepcranalysisinmyzuspersicae AT zhangyanning casestudyusingrecommendedreferencegenesactinand18sforreversetranscriptionquantitativerealtimepcranalysisinmyzuspersicae AT jianghongyun casestudyusingrecommendedreferencegenesactinand18sforreversetranscriptionquantitativerealtimepcranalysisinmyzuspersicae |