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

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.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.Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is an efficient, sensitive and reliable technique to quantify transcript expression levels. qRT-PCR is fast, easy to use and provides simultaneous measurement of gene expression in many different samples for a limited number of genes [1,2]. qRT-PCR has various applications, such as clinical diagnostics [3], analysis of tissue-specific gene expression in humans [4], and gene expression studies in plants [5]. Appropriate normalisation is very important for quantification of transcript expression lev