Abstract
BackgroundQuantitative 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.ResultsSeven 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.ConclusionsgeNorm, 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.
Highlights
Quantitative real-time reverse transcriptase polymerase chain reaction is a sensitive technique for quantifying gene expression levels
This case study illustrates the use of Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) in chicory using the fructan 1-exohydrolaseII a, 1-FEHIIa (FEHIIa), and fructan 1-exohydrolaseIIb, 1-FEHIIb (FEHIIb) genes involved in the inulin degradation pathway
The aims of this study are i) to rank the candidate reference genes according to expression stability across chicory root and leaf tissues using three different methods for expression stability measurements, ii) to develop and evaluate qRTPCR methods for these genes in chicory, iii) to select appropriate reference genes to use for normalisation of gene expression by qRT-PCR in chicory and iv) to demonstrate their usefulness in qRT-PCR by analysing the expression level of fructan 1-exohydrolaseII, 1-FEHII (FEHII) in chicory root and leaf tissues, as an example for the genes involved in inulin accumulation
Summary
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. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is an efficient, sensitive and reliable technique to quantify transcript expression levels. This case study illustrates the use of qRT-PCR for improving inulin production in chicory. Investigation of the expression level of genes encoding enzymes involved in the inulin biosynthesis and degradation pathway will enable breeders to improve inulin content. This case study illustrates the use of qRT-PCR in chicory using the fructan 1-exohydrolaseII a, 1-FEHIIa (FEHIIa), and fructan 1-exohydrolaseIIb, 1-FEHIIb (FEHIIb) genes involved in the inulin degradation pathway. The encoded enzymes are believed to catalyse fructan depolymerisation at the end of the growing season, as well as during storage and forcing of tubers and tuberous roots [16,17,18,19]
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