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Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii
Real-time quantitative reverse transcription-PCR (qRT-PCR) is an undeniably effective tool for measuring levels of gene expression, but the accuracy and reliability of the statistical data obtained depend mainly on the basal expression of selected housekeeping genes in many samples. To date, there h...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
PeerJ Inc.
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6487802/ https://www.ncbi.nlm.nih.gov/pubmed/31086748 http://dx.doi.org/10.7717/peerj.6834 |
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author | Yang, Teizhu Gu, Bingning Xu, Guolyu Shi, Yanmei Shen, Heding Rao, Rongcheng Mzuka, Hellen Lucas |
author_facet | Yang, Teizhu Gu, Bingning Xu, Guolyu Shi, Yanmei Shen, Heding Rao, Rongcheng Mzuka, Hellen Lucas |
author_sort | Yang, Teizhu |
collection | PubMed |
description | Real-time quantitative reverse transcription-PCR (qRT-PCR) is an undeniably effective tool for measuring levels of gene expression, but the accuracy and reliability of the statistical data obtained depend mainly on the basal expression of selected housekeeping genes in many samples. To date, there have been few analyses of stable housekeeping genes in Onchidium reevesii under salinity stress and injury. In this study, the gene expression stabilities of seven commonly used housekeeping genes, CYC, RPL28S, ACTB, TUBB, EF1a, Ubiq and 18S RNA, were investigated using BestKeeper, geNorm, NormFinder and RefFinfer. Although the results of the four programs varied to some extent, in general, RPL28S, TUBB, ACTB and EF1a were ranked highly. ACTB and TUBB were found to be the most stable housekeeping genes under salinity stress, and EF1a plus TUBB was the most stable combination under injury stress. When analysing target gene expression in different tissues, RPL28S or EF1a should be selected as the reference gene according to the level of target gene expression. Under extreme environmental stress (salinity) conditions, ACTB (0 ppt, 5 ppt, 15 ppt, 25 ppt) and TUBB (35 ppt) are reasonable reference gene choices when expression stability and abundance are considered. Under conditions of 15 ppt salinity and injury stress, our results showed that the best two-gene combination was TUBB plus EF1a. Therefore, we suggest that RPL28S, ACTB and TUBB are suitable reference genes for evaluating mRNA transcript levels. Based on candidate gene expression analysis, the tolerance of O. reevesii to low salinity (low osmotic pressure) is reduced compared to its tolerance to high salinity (high osmotic pressure). These findings will help researchers obtain accurate results in future quantitative gene expression analyses of O. reevesii under other stress conditions. |
format | Online Article Text |
id | pubmed-6487802 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | PeerJ Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-64878022019-05-13 Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii Yang, Teizhu Gu, Bingning Xu, Guolyu Shi, Yanmei Shen, Heding Rao, Rongcheng Mzuka, Hellen Lucas PeerJ Agricultural Science Real-time quantitative reverse transcription-PCR (qRT-PCR) is an undeniably effective tool for measuring levels of gene expression, but the accuracy and reliability of the statistical data obtained depend mainly on the basal expression of selected housekeeping genes in many samples. To date, there have been few analyses of stable housekeeping genes in Onchidium reevesii under salinity stress and injury. In this study, the gene expression stabilities of seven commonly used housekeeping genes, CYC, RPL28S, ACTB, TUBB, EF1a, Ubiq and 18S RNA, were investigated using BestKeeper, geNorm, NormFinder and RefFinfer. Although the results of the four programs varied to some extent, in general, RPL28S, TUBB, ACTB and EF1a were ranked highly. ACTB and TUBB were found to be the most stable housekeeping genes under salinity stress, and EF1a plus TUBB was the most stable combination under injury stress. When analysing target gene expression in different tissues, RPL28S or EF1a should be selected as the reference gene according to the level of target gene expression. Under extreme environmental stress (salinity) conditions, ACTB (0 ppt, 5 ppt, 15 ppt, 25 ppt) and TUBB (35 ppt) are reasonable reference gene choices when expression stability and abundance are considered. Under conditions of 15 ppt salinity and injury stress, our results showed that the best two-gene combination was TUBB plus EF1a. Therefore, we suggest that RPL28S, ACTB and TUBB are suitable reference genes for evaluating mRNA transcript levels. Based on candidate gene expression analysis, the tolerance of O. reevesii to low salinity (low osmotic pressure) is reduced compared to its tolerance to high salinity (high osmotic pressure). These findings will help researchers obtain accurate results in future quantitative gene expression analyses of O. reevesii under other stress conditions. PeerJ Inc. 2019-04-26 /pmc/articles/PMC6487802/ /pubmed/31086748 http://dx.doi.org/10.7717/peerj.6834 Text en ©2019 Yang et al. http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited. |
spellingShingle | Agricultural Science Yang, Teizhu Gu, Bingning Xu, Guolyu Shi, Yanmei Shen, Heding Rao, Rongcheng Mzuka, Hellen Lucas Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii |
title | Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii |
title_full | Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii |
title_fullStr | Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii |
title_full_unstemmed | Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii |
title_short | Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii |
title_sort | identification of candidate reference genes for qrt-pcr normalization studies of salinity stress and injury in onchidium reevesii |
topic | Agricultural Science |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6487802/ https://www.ncbi.nlm.nih.gov/pubmed/31086748 http://dx.doi.org/10.7717/peerj.6834 |
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