Abstract
Quantitative real-time PCR (qRT-PCR) is an efficient method to estimate the gene expression levels but the accuracy of its result largely depends on the stability of the reference gene. Many studies have reported considerable variation in the expression of reference genes (RGs) in different tissue and conditions. Therefore, screening for appropriate RGs with stable expression is crucial for functional analysis of the target gene. Two closely related crucifers Brassica juncea (cultivated) and Camelina sativa (wild) respond differently towards various abiotic and biotic stress where C. sativa exhibits higher tolerance to various stress. Comparative gene expression analysis of the target genes between two such species is the key approach to understand the mechanism of a plant’s response to stress. However, using an unsuitable RG can lead to misinterpretation of expression levels of the target gene in such studies. In this investigation, the stability of seven candidate RGs including traditional housekeeping genes (HKGs) and novel candidate RGs were identified across diverse sample sets of B. juncea and C. sativa representing- hormone treated, wounded, Alternaria brassicae inoculated and combination treated samples (exogenous hormone treatment followed by A. brassicae inoculation). In this investigation, we identified stable RGs in both the species and the most suitable RGs to perform an unbiased comparative gene expression analysis between B. juncea and C. sativa. Results revealed that TIPS41 and PP2A were identified as the overall best performing RGs in both the species. However, the most suitable RG for each sample subset representing different condition must be individually selected. In Hormone treated and wounded samples TIPS41 expressed stably in both the species and in A. brassicae inoculated and combination treatment performance of PP2A was the best. In this study, for the first time, we have identified and validated stable reference gene in C. sativa for accurate normalization of gene expression data.
Highlights
Quantification of mRNA transcript levels provides important insights into the intricate metabolic pathways and signaling networks underlying the plant’s physiological plant’s physiological response to various abiotic and biotic stresses. Quantitative real-time PCR (qRT-PCR) is a sensitive, accurate and costeffective method to evaluate the expression of the target gene in different tissues, organs and treatments [1,2]
Expression profiling of reference genes in B. juncea and C. sativa qRT-PCR assay based on SYBR green detection chemistry was designed for transcript profiling of seven RGs (ACT7, CAC, elongation factor 1- alpha (EF1A), PP2A, TIPS41, tubulin alpha (TUA), UBQ9) in two species
We evaluated the expression stability of 7 candidate RGs across diverse sample sets of B. juncea and C. sativa in order to identify suitable RGs for normalization of gene expression analysis in both the genotypes
Summary
Quantification of mRNA transcript levels provides important insights into the intricate metabolic pathways and signaling networks underlying the plant’s physiological plant’s physiological response to various abiotic and biotic stresses. qRT-PCR is a sensitive, accurate and costeffective method to evaluate the expression of the target gene in different tissues, organs and treatments [1,2]. QRT-PCR is a sensitive, accurate and costeffective method to evaluate the expression of the target gene in different tissues, organs and treatments [1,2]. The accuracy of the expression data is affected by many factors, such as RNA quality, purity, PCR amplification efficiency, technical and true biological variations [3,4]. To control these variables and avoid bias in qRT-PCR, selecting stably expressed RGs under different experimental conditions are crucial for normalization. RGs are the genes which maintain a constant expression level in all cell types and under every experimental condition. Selecting stably expressed RG is crucial for accurate normalization of gene expression data
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