Selection and Validation of Novel RT-qPCR Reference Genes under Hormonal Stimuli and in Different Tissues of Santalum album

Abstract

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used technique to investigate gene expression levels due to its high throughput, specificity, and sensitivity. An appropriate reference gene is essential for RT-qPCR analysis to obtain accurate and reliable results. To date, no reliable reference gene has been validated for the economically tropical tree, sandalwood (Santalum album L.). In this study, 13 candidate reference genes, including 12 novel putative reference genes selected from a large set of S. album transcriptome data, as well as the currently used β-actin gene (ACT), were validated in different tissues (stem, leaf, root and callus), as well as callus tissue under salicylic acid (SA), jasmonic acid methyl ester (MeJA), and gibberellin (GA) treatments using geNorm, NormFinder, BestKeeper, Delta Ct and comprehensive RefFinder algorithms. Several novel candidate reference genes were much more stable than the currently used traditional gene ACT. ODD paired with Fbp1 for SA treatment, CSA and Fbp3 for MeJA treatment, PP2C and Fbp2 for GA treatment, as well as Fbp1 combined with Fbp2 for the total of three hormone treatments were the most accurate reference genes, respectively. FAB1A, when combined with PP2C, was identified as the most suitable reference gene combination for the four tissues tested, while the combination of HLMt, PPR and FAB1A were the most optimal reference genes for all of the experimental samples. In addition, to verify our results, the relative expression level of the SaSSy gene was evaluated by the validated reference genes and their combinations in the three S. album tissues and under MeJA treatment. The evaluated reference genes in this study will improve the accuracy of RT-qPCR analysis and will benefit S. album functional genomics studies in different tissues and under hormone stimuli in the future.

Selection and validation of reference genes for measuring gene expression in Toona ciliata under different experimental conditions by quantitative real-time PCR analysis

“…The threshold used was 0.15 phosphatase in plants [49]. Although it not often has been used as a candidate internal reference gene, it is stable in our study under pest stress and in different tissues for GA treatment of Santalum album [50]. Therefore, when screening reference genes in other species, PP2Cs can be considered as a candidate reference gene.…”

“…Protein phosphatase can reverse the phosphorylation of protein kinases, thereby dynamically controlling protein phosphorylation and protein phosphatase 2Cs ( PP2Cs ) is the most abundant type of phosphatase in plants [ 49 ]. Although it not often has been used as a candidate internal reference gene, it is stable in our study under pest stress and in different tissues for GA treatment of Santalum album [ 50 ]. Therefore, when screening reference genes in other species, PP2Cs can be considered as a candidate reference gene.…”

Validation of suitable genes for normalization of diurnal gene expression studies inChenopodium quinoa

“…Several studies have identified negligible to major differences in the stability rankings of the same gene. RefFinder and RankAgreeg are commonly used tools to configure a standard ranking for reference genes [3,5,11]. Here, we introduced a novel and straightforward method to find suitable reference genes for normalization of diurnal expression data.…”

Genome-Wide Characterization, Expression Profile Analysis of WRKY Family Genes in Santalum album and Functional Identification of Their Role in Abiotic Stress

“…After 40 cycles, melting curve analysis was performed ranging from 60 to 95 °C. The internal reference genes used in different tissues and phytohormone treatments followed Yan et al [57], i.e., FAB1A and PP2C for four tissues, ODD and Fbp1 for the SA treatment, as well as CSA and Fbp3 for the MeJA treatment. The relative gene expression level was calculated using the 2 −ΔΔ C q method.…”