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Selection of reference genes for diurnal and developmental time-course real-time PCR expression analyses in lettuce
BACKGROUND: Real-time quantitative polymerase chain reaction (RT-qPCR) analysis is a low cost and sensitive technique that is widely used to measure levels of gene expression. Selecting and validating appropriate reference genes for normalising target gene expression should be the first step in any...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4804537/ https://www.ncbi.nlm.nih.gov/pubmed/27011764 http://dx.doi.org/10.1186/s13007-016-0121-y |
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author | Sgamma, Tiziana Pape, Judith Massiah, Andrea Jackson, Stephen |
author_facet | Sgamma, Tiziana Pape, Judith Massiah, Andrea Jackson, Stephen |
author_sort | Sgamma, Tiziana |
collection | PubMed |
description | BACKGROUND: Real-time quantitative polymerase chain reaction (RT-qPCR) analysis is a low cost and sensitive technique that is widely used to measure levels of gene expression. Selecting and validating appropriate reference genes for normalising target gene expression should be the first step in any expression study to avoid inaccurate results. RESULTS: In this study, ten candidate genes were tested for their suitability for use as reference genes in diurnal and developmental timecourse experiments in lettuce. The candidate reference genes were then used to normalise the expression pattern of the FLOWERING LOCUS T (FT) gene, one of key genes involved in the flowering time pathway whose expression is known to vary throughout the day and at different stages of development. Three reference genes, LsPP2A-1 (PROTEIN PHOSPHATASE 2A-1), LsPP2AA3 (PROTEIN PHOSPHATASE 2A REGULATORY SUBUNIT A3) and LsTIP41 (TAP42-INTERACTING PROTEIN OF 41 kDa), were the most stably expressed candidate reference genes throughout both the diurnal and developmental timecourse experiments. In the developmental experiment using just LsPP2A-1 and LsTIP41 as reference genes would be sufficient for accurate normalisation, whilst in the diurnal experiment all three reference genes, LsPP2A-1, LsPP2AA3 and LsTIP41, would be necessary. The FT expression pattern obtained demonstrates that the use of multiple and robust reference genes for RT-qPCR expression analyses results in a more accurate and reliable expression profile. CONCLUSIONS: Reference genes suitable for use in diurnal and developmental timecourse experiments in lettuce were identified and used to produce a more accurate and reliable analysis of lsFT expression levels than previously obtained in such timecourse experiments. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13007-016-0121-y) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-4804537 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-48045372016-03-23 Selection of reference genes for diurnal and developmental time-course real-time PCR expression analyses in lettuce Sgamma, Tiziana Pape, Judith Massiah, Andrea Jackson, Stephen Plant Methods Methodology BACKGROUND: Real-time quantitative polymerase chain reaction (RT-qPCR) analysis is a low cost and sensitive technique that is widely used to measure levels of gene expression. Selecting and validating appropriate reference genes for normalising target gene expression should be the first step in any expression study to avoid inaccurate results. RESULTS: In this study, ten candidate genes were tested for their suitability for use as reference genes in diurnal and developmental timecourse experiments in lettuce. The candidate reference genes were then used to normalise the expression pattern of the FLOWERING LOCUS T (FT) gene, one of key genes involved in the flowering time pathway whose expression is known to vary throughout the day and at different stages of development. Three reference genes, LsPP2A-1 (PROTEIN PHOSPHATASE 2A-1), LsPP2AA3 (PROTEIN PHOSPHATASE 2A REGULATORY SUBUNIT A3) and LsTIP41 (TAP42-INTERACTING PROTEIN OF 41 kDa), were the most stably expressed candidate reference genes throughout both the diurnal and developmental timecourse experiments. In the developmental experiment using just LsPP2A-1 and LsTIP41 as reference genes would be sufficient for accurate normalisation, whilst in the diurnal experiment all three reference genes, LsPP2A-1, LsPP2AA3 and LsTIP41, would be necessary. The FT expression pattern obtained demonstrates that the use of multiple and robust reference genes for RT-qPCR expression analyses results in a more accurate and reliable expression profile. CONCLUSIONS: Reference genes suitable for use in diurnal and developmental timecourse experiments in lettuce were identified and used to produce a more accurate and reliable analysis of lsFT expression levels than previously obtained in such timecourse experiments. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13007-016-0121-y) contains supplementary material, which is available to authorized users. BioMed Central 2016-03-22 /pmc/articles/PMC4804537/ /pubmed/27011764 http://dx.doi.org/10.1186/s13007-016-0121-y Text en © Sgamma et al. 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Sgamma, Tiziana Pape, Judith Massiah, Andrea Jackson, Stephen Selection of reference genes for diurnal and developmental time-course real-time PCR expression analyses in lettuce |
title | Selection of reference genes for diurnal and developmental time-course real-time PCR expression analyses in lettuce |
title_full | Selection of reference genes for diurnal and developmental time-course real-time PCR expression analyses in lettuce |
title_fullStr | Selection of reference genes for diurnal and developmental time-course real-time PCR expression analyses in lettuce |
title_full_unstemmed | Selection of reference genes for diurnal and developmental time-course real-time PCR expression analyses in lettuce |
title_short | Selection of reference genes for diurnal and developmental time-course real-time PCR expression analyses in lettuce |
title_sort | selection of reference genes for diurnal and developmental time-course real-time pcr expression analyses in lettuce |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4804537/ https://www.ncbi.nlm.nih.gov/pubmed/27011764 http://dx.doi.org/10.1186/s13007-016-0121-y |
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