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Open Access Highly Accessed Research article

Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR

Asad Maroufi12, Erik Van Bockstaele13 and Marc De Loose24*

Author Affiliations

1 Department of Plant Production, Faculty of Bioscience Engineering, Coupure Links 653, Gent University, 9000 Ghent, Belgium

2 Institute for Agricultural and Fisheries Research (ILVO), Burg Van Gansberghelaan 115 bus 1, 9820 Merelbeke, Belgium

3 Institute for Agricultural and Fisheries Research (ILVO), Burg Van Gansberghelaan 96 bus 1, 9820 Merelbeke, Belgium

4 Department of Plant Biotechnology and Genetics, Faculty of Sciences, Gent University, KL Ledeganckstraat 35, 9000 Ghent, Belgium

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BMC Molecular Biology 2010, 11:15  doi:10.1186/1471-2199-11-15

Published: 15 February 2010

Abstract

Background

Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a sensitive technique for quantifying gene expression levels. One or more appropriate reference genes must be selected to accurately compare mRNA transcripts across different samples and tissues. Thus far, only actin-2 has been used as a reference gene for qRT-PCR in chicory, and a full comparison of several candidate reference genes in chicory has not yet been reported.

Results

Seven candidate reference genes, including nicotinamide adenine dinucleotide dehydrogenase (NADHD), actin (ACT), β-tubulin (TUB), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), histone H3 (H3), elongation factor 1-alpha (EF) and 18S rRNA (rRNA) were selected to study the expression stability for normalisation of gene expression in chicory. Primer specificity and amplification efficiency were verified for each gene. The expression stability of these genes was analysed across chicory root and leaf tissues using geNorm, NormFinder and BestKeeper software. ACT, EF, and rRNA were the most stable genes as identified by the three different analysis methods. In addition, the use of ACT, EF and GAPDH as reference genes was illustrated by analysing 1-FEHII (FEHII) expression in chicory root and leaf tissues. These analyses revealed the biological variation in FEHII transcript expression among the tissues studied, and between individual plants.

Conclusions

geNorm, NormFinder, and BestKeeper analyses indicated that ACT, EF and rRNA had the highest expression stability across leaf and root tissues, while GAPDH and NADHD showed relatively low expression stability. The results of this study emphasise the importance of validating reference genes for qRT-PCR analysis in chicory. The use of the most stable reference genes such as ACT and EF allows accurate normalisation of gene expression in chicory leaf and root tissues.