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Identification of a Novel Reference Gene for Apple Transcriptional Profiling under Postharvest Conditions
Reverse Transcription quantitative PCR (RT-qPCR) is one of the most important techniques for gene expression profiling due to its high sensibility and reproducibility. However, the reliability of the results is highly dependent on data normalization, performed by comparisons between the expression p...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361542/ https://www.ncbi.nlm.nih.gov/pubmed/25774904 http://dx.doi.org/10.1371/journal.pone.0120599 |
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author | Storch, Tatiane Timm Pegoraro, Camila Finatto, Taciane Quecini, Vera Rombaldi, Cesar Valmor Girardi, César Luis |
author_facet | Storch, Tatiane Timm Pegoraro, Camila Finatto, Taciane Quecini, Vera Rombaldi, Cesar Valmor Girardi, César Luis |
author_sort | Storch, Tatiane Timm |
collection | PubMed |
description | Reverse Transcription quantitative PCR (RT-qPCR) is one of the most important techniques for gene expression profiling due to its high sensibility and reproducibility. However, the reliability of the results is highly dependent on data normalization, performed by comparisons between the expression profiles of the genes of interest against those of constitutively expressed, reference genes. Although the technique is widely used in fruit postharvest experiments, the transcription stability of reference genes has not been thoroughly investigated under these experimental conditions. Thus, we have determined the transcriptional profile, under these conditions, of three genes commonly used as reference—ACTIN (MdACT), PROTEIN DISULPHIDE ISOMERASE (MdPDI) and UBIQUITIN-CONJUGATING ENZYME E2 (MdUBC)—along with two novel candidates—HISTONE 1 (MdH1) and NUCLEOSSOME ASSEMBLY 1 PROTEIN (MdNAP1). The expression profile of the genes was investigated throughout five experiments, with three of them encompassing the postharvest period and the other two, consisting of developmental and spatial phases. The transcriptional stability was comparatively investigated using four distinct software packages: BestKeeper, NormFinder, geNorm and DataAssist. Gene ranking results for transcriptional stability were similar for the investigated software packages, with the exception of BestKeeper. The classic reference gene MdUBC ranked among the most stably transcribed in all investigated experimental conditions. Transcript accumulation profiles for the novel reference candidate gene MdH1 were stable throughout the tested conditions, especially in experiments encompassing the postharvest period. Thus, our results present a novel reference gene for postharvest experiments in apple and reinforce the importance of checking the transcription profile of reference genes under the experimental conditions of interest. |
format | Online Article Text |
id | pubmed-4361542 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-43615422015-03-23 Identification of a Novel Reference Gene for Apple Transcriptional Profiling under Postharvest Conditions Storch, Tatiane Timm Pegoraro, Camila Finatto, Taciane Quecini, Vera Rombaldi, Cesar Valmor Girardi, César Luis PLoS One Research Article Reverse Transcription quantitative PCR (RT-qPCR) is one of the most important techniques for gene expression profiling due to its high sensibility and reproducibility. However, the reliability of the results is highly dependent on data normalization, performed by comparisons between the expression profiles of the genes of interest against those of constitutively expressed, reference genes. Although the technique is widely used in fruit postharvest experiments, the transcription stability of reference genes has not been thoroughly investigated under these experimental conditions. Thus, we have determined the transcriptional profile, under these conditions, of three genes commonly used as reference—ACTIN (MdACT), PROTEIN DISULPHIDE ISOMERASE (MdPDI) and UBIQUITIN-CONJUGATING ENZYME E2 (MdUBC)—along with two novel candidates—HISTONE 1 (MdH1) and NUCLEOSSOME ASSEMBLY 1 PROTEIN (MdNAP1). The expression profile of the genes was investigated throughout five experiments, with three of them encompassing the postharvest period and the other two, consisting of developmental and spatial phases. The transcriptional stability was comparatively investigated using four distinct software packages: BestKeeper, NormFinder, geNorm and DataAssist. Gene ranking results for transcriptional stability were similar for the investigated software packages, with the exception of BestKeeper. The classic reference gene MdUBC ranked among the most stably transcribed in all investigated experimental conditions. Transcript accumulation profiles for the novel reference candidate gene MdH1 were stable throughout the tested conditions, especially in experiments encompassing the postharvest period. Thus, our results present a novel reference gene for postharvest experiments in apple and reinforce the importance of checking the transcription profile of reference genes under the experimental conditions of interest. Public Library of Science 2015-03-16 /pmc/articles/PMC4361542/ /pubmed/25774904 http://dx.doi.org/10.1371/journal.pone.0120599 Text en © 2015 Storch 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, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Storch, Tatiane Timm Pegoraro, Camila Finatto, Taciane Quecini, Vera Rombaldi, Cesar Valmor Girardi, César Luis Identification of a Novel Reference Gene for Apple Transcriptional Profiling under Postharvest Conditions |
title | Identification of a Novel Reference Gene for Apple Transcriptional Profiling under Postharvest Conditions |
title_full | Identification of a Novel Reference Gene for Apple Transcriptional Profiling under Postharvest Conditions |
title_fullStr | Identification of a Novel Reference Gene for Apple Transcriptional Profiling under Postharvest Conditions |
title_full_unstemmed | Identification of a Novel Reference Gene for Apple Transcriptional Profiling under Postharvest Conditions |
title_short | Identification of a Novel Reference Gene for Apple Transcriptional Profiling under Postharvest Conditions |
title_sort | identification of a novel reference gene for apple transcriptional profiling under postharvest conditions |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361542/ https://www.ncbi.nlm.nih.gov/pubmed/25774904 http://dx.doi.org/10.1371/journal.pone.0120599 |
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