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Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative
Asad Maroufi1, Erik Van Bockstaele, Marc De Loose
1Department of Plant Production, Faculty of Bioscience Engineering, Coupure Links 653, Gent University, 9000 Ghent, Belgium.
BMC Molecular Biology
|February 17, 2010
Summary
Selecting stable reference genes like actin (ACT) and elongation factor 1-alpha (EF) is crucial for accurate gene expression analysis in chicory. This study identified ACT, EF, and 18S rRNA (rRNA) as the most reliable reference genes for qRT-PCR in chicory tissues.
Area of Science:
- Plant molecular biology
- Gene expression analysis
Background:
- Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) requires stable reference genes for accurate gene expression quantification.
- Previously, only actin-2 was used in chicory, lacking comprehensive validation of multiple candidates.
Purpose of the Study:
- To evaluate the expression stability of seven candidate reference genes in chicory.
- To identify the most reliable reference genes for normalizing qRT-PCR data in chicory leaf and root tissues.
Main Methods:
- Seven candidate reference genes (NADHD, ACT, TUB, GADPH, H3, EF, rRNA) were selected and validated for primer specificity and amplification efficiency.
- Expression stability was analyzed across chicory root and leaf tissues using geNorm, NormFinder, and BestKeeper software.
- The utility of selected reference genes was demonstrated by analyzing 1-FEHII (FEHII) gene expression.
Main Results:
- Actin (ACT), elongation factor 1-alpha (EF), and 18S rRNA (rRNA) exhibited the highest expression stability across both tissues.
- Nicotinamide adenine dinucleotide dehydrogenase (NADHD) and glyceraldehyde-3-phosphate-dehydrogenase (GADPH) showed lower stability.
- Analysis of FEHII expression confirmed the biological variation and the importance of appropriate reference gene selection.
Conclusions:
- ACT, EF, and rRNA are recommended as the most stable reference genes for qRT-PCR in chicory leaf and root tissues.
- The study highlights the critical need for reference gene validation in chicory gene expression studies.
- Utilizing validated stable reference genes like ACT and EF ensures accurate normalization and reliable results.
