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Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus
Quantitative real-time reverse transcription PCR (RT-qPCR) has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
PeerJ Inc.
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5408727/ https://www.ncbi.nlm.nih.gov/pubmed/28462052 http://dx.doi.org/10.7717/peerj.3260 |
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author | Wang, Kai Niu, Yi Wang, Qijun Liu, Haili Jin, Yi Zhang, Shenglin |
author_facet | Wang, Kai Niu, Yi Wang, Qijun Liu, Haili Jin, Yi Zhang, Shenglin |
author_sort | Wang, Kai |
collection | PubMed |
description | Quantitative real-time reverse transcription PCR (RT-qPCR) has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes. Amorphophallus is a perennial plant with a high content of konjac glucomannan (KGM) in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes in Amorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genes Amorphophallus were cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging) in A. albus and A. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated that EF1-a, EIF4A, H3 and UBQ were the best reference genes under heat stress in Amorphophallus. Furthermore, EF1-a, EIF4A, TUB, and RP were the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined that EF1-α, EIF4A, and CYP were stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock protein SHSP, which is related to heat stress in Amorphophallus. In sum, EF1-α and EIF4A were the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different biological processes to normalize gene expression. Our results will provide researchers with appropriate reference genes for further gene expression quantification using RT-qPCR in Amorphophallus. |
format | Online Article Text |
id | pubmed-5408727 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | PeerJ Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-54087272017-05-01 Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus Wang, Kai Niu, Yi Wang, Qijun Liu, Haili Jin, Yi Zhang, Shenglin PeerJ Agricultural Science Quantitative real-time reverse transcription PCR (RT-qPCR) has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes. Amorphophallus is a perennial plant with a high content of konjac glucomannan (KGM) in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes in Amorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genes Amorphophallus were cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging) in A. albus and A. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated that EF1-a, EIF4A, H3 and UBQ were the best reference genes under heat stress in Amorphophallus. Furthermore, EF1-a, EIF4A, TUB, and RP were the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined that EF1-α, EIF4A, and CYP were stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock protein SHSP, which is related to heat stress in Amorphophallus. In sum, EF1-α and EIF4A were the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different biological processes to normalize gene expression. Our results will provide researchers with appropriate reference genes for further gene expression quantification using RT-qPCR in Amorphophallus. PeerJ Inc. 2017-04-26 /pmc/articles/PMC5408727/ /pubmed/28462052 http://dx.doi.org/10.7717/peerj.3260 Text en ©2017 Wang 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 Wang, Kai Niu, Yi Wang, Qijun Liu, Haili Jin, Yi Zhang, Shenglin Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus |
title | Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus |
title_full | Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus |
title_fullStr | Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus |
title_full_unstemmed | Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus |
title_short | Cloning and evaluation of reference genes for quantitative real-time PCR analysis in Amorphophallus |
title_sort | cloning and evaluation of reference genes for quantitative real-time pcr analysis in amorphophallus |
topic | Agricultural Science |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5408727/ https://www.ncbi.nlm.nih.gov/pubmed/28462052 http://dx.doi.org/10.7717/peerj.3260 |
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