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The selection and validation of reference genes for quantitative real-time PCR studies in near-isogenic susceptible and resistant tomato lines, infected with the geminivirus tomato curly stunt virus
Quantitative real-time PCR (qPCR) is a sensitive and commonly used technique for gene expression profiling and provides insight into biological systems. Successful qPCR requires the use of appropriate reference genes for the normalization of data. In the present study, we aimed to identify and asses...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Public Library of Science
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10374155/ https://www.ncbi.nlm.nih.gov/pubmed/37498814 http://dx.doi.org/10.1371/journal.pone.0284456 |
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author | Bokhale, Mamokete Mwaba, Imanu Allie, Farhahna |
author_facet | Bokhale, Mamokete Mwaba, Imanu Allie, Farhahna |
author_sort | Bokhale, Mamokete |
collection | PubMed |
description | Quantitative real-time PCR (qPCR) is a sensitive and commonly used technique for gene expression profiling and provides insight into biological systems. Successful qPCR requires the use of appropriate reference genes for the normalization of data. In the present study, we aimed to identify and assess the best-suited reference genes in near-isogenic resistant (R) and susceptible (S) tomato lines infected with begomovirus Tomato curly stunt virus (ToCSV). Ten candidate reference genes namely, Actin7 (ACT), β-6 Tubulin (TUB), Ubiquitin 3 (UBI), Clathrin adaptor complexes medium subunit (CAC), Phytoene desaturase (PDS), Expressed protein (EXP), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Adenine phosphoribosyl transferase-like protein (APT1), TAP42-interacting protein (TIP41) and Elongation factor 1-alpha (EF1α) were selected and evaluated for their expression stability in resistant and susceptible tomato leaves using the analytical tools geNorm, NormFinder, BestKeeper, and RefFinder. After ranking the reference genes from most to least stable, the results suggested that a combination of ACT, EXP, and EF1α in the S lines and a combination of TIP41, APT1, and ACT in the R line is appropriate for qPCR normalization. Furthermore, to validate the identified reference genes, iron superoxide dismutase (SOD), heat shock protein 70 (HSP70) and Glutathione-S-transferase (GST) were selected as targets for normalization. The relative expression of the target genes varied when normalized against the most stable reference genes in comparison to the least stable genes. These results highlight the importance of careful selection of reference genes for accurate normalization in qPCR studies. |
format | Online Article Text |
id | pubmed-10374155 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-103741552023-07-28 The selection and validation of reference genes for quantitative real-time PCR studies in near-isogenic susceptible and resistant tomato lines, infected with the geminivirus tomato curly stunt virus Bokhale, Mamokete Mwaba, Imanu Allie, Farhahna PLoS One Research Article Quantitative real-time PCR (qPCR) is a sensitive and commonly used technique for gene expression profiling and provides insight into biological systems. Successful qPCR requires the use of appropriate reference genes for the normalization of data. In the present study, we aimed to identify and assess the best-suited reference genes in near-isogenic resistant (R) and susceptible (S) tomato lines infected with begomovirus Tomato curly stunt virus (ToCSV). Ten candidate reference genes namely, Actin7 (ACT), β-6 Tubulin (TUB), Ubiquitin 3 (UBI), Clathrin adaptor complexes medium subunit (CAC), Phytoene desaturase (PDS), Expressed protein (EXP), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Adenine phosphoribosyl transferase-like protein (APT1), TAP42-interacting protein (TIP41) and Elongation factor 1-alpha (EF1α) were selected and evaluated for their expression stability in resistant and susceptible tomato leaves using the analytical tools geNorm, NormFinder, BestKeeper, and RefFinder. After ranking the reference genes from most to least stable, the results suggested that a combination of ACT, EXP, and EF1α in the S lines and a combination of TIP41, APT1, and ACT in the R line is appropriate for qPCR normalization. Furthermore, to validate the identified reference genes, iron superoxide dismutase (SOD), heat shock protein 70 (HSP70) and Glutathione-S-transferase (GST) were selected as targets for normalization. The relative expression of the target genes varied when normalized against the most stable reference genes in comparison to the least stable genes. These results highlight the importance of careful selection of reference genes for accurate normalization in qPCR studies. Public Library of Science 2023-07-27 /pmc/articles/PMC10374155/ /pubmed/37498814 http://dx.doi.org/10.1371/journal.pone.0284456 Text en © 2023 Bokhale 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 Bokhale, Mamokete Mwaba, Imanu Allie, Farhahna The selection and validation of reference genes for quantitative real-time PCR studies in near-isogenic susceptible and resistant tomato lines, infected with the geminivirus tomato curly stunt virus |
title | The selection and validation of reference genes for quantitative real-time PCR studies in near-isogenic susceptible and resistant tomato lines, infected with the geminivirus tomato curly stunt virus |
title_full | The selection and validation of reference genes for quantitative real-time PCR studies in near-isogenic susceptible and resistant tomato lines, infected with the geminivirus tomato curly stunt virus |
title_fullStr | The selection and validation of reference genes for quantitative real-time PCR studies in near-isogenic susceptible and resistant tomato lines, infected with the geminivirus tomato curly stunt virus |
title_full_unstemmed | The selection and validation of reference genes for quantitative real-time PCR studies in near-isogenic susceptible and resistant tomato lines, infected with the geminivirus tomato curly stunt virus |
title_short | The selection and validation of reference genes for quantitative real-time PCR studies in near-isogenic susceptible and resistant tomato lines, infected with the geminivirus tomato curly stunt virus |
title_sort | selection and validation of reference genes for quantitative real-time pcr studies in near-isogenic susceptible and resistant tomato lines, infected with the geminivirus tomato curly stunt virus |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10374155/ https://www.ncbi.nlm.nih.gov/pubmed/37498814 http://dx.doi.org/10.1371/journal.pone.0284456 |
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