Analysis of qPCR reference gene stability determination methods and a practical approach for efficiency calculation on a turbot (Scophthalmus maximus) gonad dataset
[Background] Gene expression analysis by reverse transcription quantitative PCR (qPCR) is the most widely used method for analyzing the expression of a moderate number of genes and also for the validation of microarray results. Several issues are crucial for a successful qPCR study, particularly the...
Published in: | BMC Genomics |
---|---|
Main Authors: | , , , , , , |
Other Authors: | , |
Format: | Article in Journal/Newspaper |
Language: | unknown |
Published: |
BioMed Central
2014
|
Subjects: | |
Online Access: | http://hdl.handle.net/10261/125891 https://doi.org/10.1186/1471-2164-15-648 https://doi.org/10.13039/501100004837 https://doi.org/10.13039/501100003176 |
id |
ftcsic:oai:digital.csic.es:10261/125891 |
---|---|
record_format |
openpolar |
spelling |
ftcsic:oai:digital.csic.es:10261/125891 2024-02-11T10:08:26+01:00 Analysis of qPCR reference gene stability determination methods and a practical approach for efficiency calculation on a turbot (Scophthalmus maximus) gonad dataset Robledo, Diego Hernández-Urcera, Jorge Cal, Rosa Pardo, Belén G. Sánchez, Laura Martínez, Paulino Viñas, Ana Ministerio de Ciencia e Innovación (España) Ministerio de Educación, Cultura y Deporte (España) 2014-08-04 http://hdl.handle.net/10261/125891 https://doi.org/10.1186/1471-2164-15-648 https://doi.org/10.13039/501100004837 https://doi.org/10.13039/501100003176 unknown BioMed Central Publisher's version http://dx.doi.org/10.1186/1471-2164-15-648 Sí BMC Genomics 15(1): 648 (2014) 1471-2164 http://hdl.handle.net/10261/125891 doi:10.1186/1471-2164-15-648 http://dx.doi.org/10.13039/501100004837 http://dx.doi.org/10.13039/501100003176 25091330 open qPCR Reference genes Amplification efficiency Turbot Scophthalmus maximus artículo http://purl.org/coar/resource_type/c_6501 2014 ftcsic https://doi.org/10.1186/1471-2164-15-64810.13039/50110000483710.13039/501100003176 2024-01-16T10:12:16Z [Background] Gene expression analysis by reverse transcription quantitative PCR (qPCR) is the most widely used method for analyzing the expression of a moderate number of genes and also for the validation of microarray results. Several issues are crucial for a successful qPCR study, particularly the selection of internal reference genes for normalization and efficiency determination. There is no agreement on which method is the best to detect the most stable genes neither on how to perform efficiency determination. In this study we offer a comprehensive evaluation of the characteristics of reference gene selection methods and how to decide which one is more reliable when they show discordant outcomes. Also, we analyze the current efficiency calculation controversy. Our dataset is composed by gonad samples of turbot at different development times reared at different temperatures. Turbot (Scophthalmus maximus) is a relevant marine aquaculture European species with increasing production in the incoming years. Since females largely outgrow males, identification of genes related to sex determination, gonad development and reproductive behavior, and analysis of their expression profiles are of primary importance for turbot industry. [Results] We analyzed gene stability of six reference genes: RPS4, RPL17, GAPDH, ACTB, UBQ and B2M using the comparative delta-CT method, Bestkeeper, NormFinder and GeNorm approaches in gonad samples of turbot. Supported by descriptive statistics, we found NormFinder to be the best method, while on the other side, GeNorm results proved to be unreliable. According to our analysis, UBQ and RPS4 were the most stable genes, while B2M was the least stable gene. We also analyzed the efficiency calculation softwares LinRegPCR, LREanalyzer, DART and PCR-Miner and we recommend LinRegPCR for research purposes since it does not systematically overestimate efficiency. [Conclusion] Our results indicate that NormFinder and LinRegPCR are the best approaches for reference gene selection and efficiency ... Article in Journal/Newspaper Scophthalmus maximus Turbot Digital.CSIC (Spanish National Research Council) BMC Genomics 15 1 648 |
institution |
Open Polar |
collection |
Digital.CSIC (Spanish National Research Council) |
op_collection_id |
ftcsic |
language |
unknown |
topic |
qPCR Reference genes Amplification efficiency Turbot Scophthalmus maximus |
spellingShingle |
qPCR Reference genes Amplification efficiency Turbot Scophthalmus maximus Robledo, Diego Hernández-Urcera, Jorge Cal, Rosa Pardo, Belén G. Sánchez, Laura Martínez, Paulino Viñas, Ana Analysis of qPCR reference gene stability determination methods and a practical approach for efficiency calculation on a turbot (Scophthalmus maximus) gonad dataset |
topic_facet |
qPCR Reference genes Amplification efficiency Turbot Scophthalmus maximus |
description |
[Background] Gene expression analysis by reverse transcription quantitative PCR (qPCR) is the most widely used method for analyzing the expression of a moderate number of genes and also for the validation of microarray results. Several issues are crucial for a successful qPCR study, particularly the selection of internal reference genes for normalization and efficiency determination. There is no agreement on which method is the best to detect the most stable genes neither on how to perform efficiency determination. In this study we offer a comprehensive evaluation of the characteristics of reference gene selection methods and how to decide which one is more reliable when they show discordant outcomes. Also, we analyze the current efficiency calculation controversy. Our dataset is composed by gonad samples of turbot at different development times reared at different temperatures. Turbot (Scophthalmus maximus) is a relevant marine aquaculture European species with increasing production in the incoming years. Since females largely outgrow males, identification of genes related to sex determination, gonad development and reproductive behavior, and analysis of their expression profiles are of primary importance for turbot industry. [Results] We analyzed gene stability of six reference genes: RPS4, RPL17, GAPDH, ACTB, UBQ and B2M using the comparative delta-CT method, Bestkeeper, NormFinder and GeNorm approaches in gonad samples of turbot. Supported by descriptive statistics, we found NormFinder to be the best method, while on the other side, GeNorm results proved to be unreliable. According to our analysis, UBQ and RPS4 were the most stable genes, while B2M was the least stable gene. We also analyzed the efficiency calculation softwares LinRegPCR, LREanalyzer, DART and PCR-Miner and we recommend LinRegPCR for research purposes since it does not systematically overestimate efficiency. [Conclusion] Our results indicate that NormFinder and LinRegPCR are the best approaches for reference gene selection and efficiency ... |
author2 |
Ministerio de Ciencia e Innovación (España) Ministerio de Educación, Cultura y Deporte (España) |
format |
Article in Journal/Newspaper |
author |
Robledo, Diego Hernández-Urcera, Jorge Cal, Rosa Pardo, Belén G. Sánchez, Laura Martínez, Paulino Viñas, Ana |
author_facet |
Robledo, Diego Hernández-Urcera, Jorge Cal, Rosa Pardo, Belén G. Sánchez, Laura Martínez, Paulino Viñas, Ana |
author_sort |
Robledo, Diego |
title |
Analysis of qPCR reference gene stability determination methods and a practical approach for efficiency calculation on a turbot (Scophthalmus maximus) gonad dataset |
title_short |
Analysis of qPCR reference gene stability determination methods and a practical approach for efficiency calculation on a turbot (Scophthalmus maximus) gonad dataset |
title_full |
Analysis of qPCR reference gene stability determination methods and a practical approach for efficiency calculation on a turbot (Scophthalmus maximus) gonad dataset |
title_fullStr |
Analysis of qPCR reference gene stability determination methods and a practical approach for efficiency calculation on a turbot (Scophthalmus maximus) gonad dataset |
title_full_unstemmed |
Analysis of qPCR reference gene stability determination methods and a practical approach for efficiency calculation on a turbot (Scophthalmus maximus) gonad dataset |
title_sort |
analysis of qpcr reference gene stability determination methods and a practical approach for efficiency calculation on a turbot (scophthalmus maximus) gonad dataset |
publisher |
BioMed Central |
publishDate |
2014 |
url |
http://hdl.handle.net/10261/125891 https://doi.org/10.1186/1471-2164-15-648 https://doi.org/10.13039/501100004837 https://doi.org/10.13039/501100003176 |
genre |
Scophthalmus maximus Turbot |
genre_facet |
Scophthalmus maximus Turbot |
op_relation |
Publisher's version http://dx.doi.org/10.1186/1471-2164-15-648 Sí BMC Genomics 15(1): 648 (2014) 1471-2164 http://hdl.handle.net/10261/125891 doi:10.1186/1471-2164-15-648 http://dx.doi.org/10.13039/501100004837 http://dx.doi.org/10.13039/501100003176 25091330 |
op_rights |
open |
op_doi |
https://doi.org/10.1186/1471-2164-15-64810.13039/50110000483710.13039/501100003176 |
container_title |
BMC Genomics |
container_volume |
15 |
container_issue |
1 |
container_start_page |
648 |
_version_ |
1790607751950368768 |