Systematic Identification of Suitable Reference Genes for Quantitative Real-Time PCR Analysis in Melissa officinalis L

Abstract

Melissa officinalis L. is well known for its lemon-scented aroma and various pharmacological properties. Despite these valuable properties, the genes involved in the biosynthetic pathways in M. officinalis are not yet well-explored when compared to other members of the mint family. For that, gene expression studies using quantitative real-time PCR (qRT-PCR) are an excellent tool. Although qRT-PCR can provide accurate results, its accuracy is highly reliant on the expression and stability of the reference gene used for normalization. Hence, selecting a suitable experiment-specific reference gene is very crucial to obtain accurate results. However, to date, there are no reports for experiment-specific reference genes in M. officinalis. Therefore, in the current study, ten commonly used reference genes were assessed for their suitability as optimal reference genes in M. officinalis under various abiotic stress conditions and different plant organs. The candidate genes were ranked based on BestKeeper, comparative ΔCt, geNorm, NormFinder, and RefFinder. Based on the results, we recommend the combination of EF-1α and GAPDH as the best reference genes to normalize gene expression studies in M. officinalis. On the contrary, HLH71 was identified as the least-performing gene. Thereafter, the reliability of the optimal gene combination was assessed by evaluating the relative gene expression of the phenylalanine ammonia lyase (PAL) gene under two elicitor treatments (gibberellic acid and jasmonic acid). PAL is a crucial gene involved directly or indirectly in the production of various economically important secondary metabolites in plants. Suitable reference genes for each experimental condition are also discussed. The findings of the current study form a basis for current and future gene expression studies in M. officinalis and other related species.

Physiological and Molecular Responses of Pyrus pyraster Seedlings to Salt Treatment Analyzed by miRNA and Cytochrome P450 Gene-Based Markers

“…qRT-PCR is a very sensitive and fully reproducible molecular technique. It provides a wide range of quantification strategies that allow the analysis of individual parts of multiplex gene expression [ 73 , 74 , 75 , 76 , 77 ]. A first step in precise and reproducible gene expression quantification is normalization to the expression levels of the verified target reference gene/s that should be proven to have only minor differences in their expressions in various developmental stages, tissue or organ types, or experimental conditions [ 77 , 78 ].…”

“…It provides a wide range of quantification strategies that allow the analysis of individual parts of multiplex gene expression [ 73 , 74 , 75 , 76 , 77 ]. A first step in precise and reproducible gene expression quantification is normalization to the expression levels of the verified target reference gene/s that should be proven to have only minor differences in their expressions in various developmental stages, tissue or organ types, or experimental conditions [ 77 , 78 ]. Many different reference genes have been tested to be suitable for qRT-PCR analysis in pears, such as GAPDH, EF1a, TUB, ACT, GAPC, SKD1, UBQ5, and YLS8 [ 44 , 79 , 80 , 81 ].…”