I Choose You: Selecting Accurate Reference Genes for qPCR Expression Analysis in Reproductive Tissues in Arabidopsis thaliana

Abstract

Quantitative real-time polymerase chain reaction (qPCR) is a widely used method to analyse the gene expression pattern in the reproductive tissues along with detecting gene levels in mutant backgrounds. This technique requires stable reference genes to normalise the expression level of target genes. Nonetheless, a considerable number of publications continue to present qPCR results normalised to a single reference gene and, to our knowledge, no comparative evaluation of multiple reference genes has been carried out in specific reproductive tissues of Arabidopsis thaliana. Herein, we assessed the expression stability levels of ten candidate reference genes (UBC9, ACT7, GAPC-2, RCE1, PP2AA3, TUA2, SAC52, YLS8, SAMDC and HIS3.3) in two conditional sets: one across flower development and the other using inflorescences from different genotypes. The stability analysis was performed using the RefFinder tool, which combines four statistical algorithms (geNorm, NormFinder, BestKeeper and the comparative ΔCt method). Our results showed that RCE1, SAC52 and TUA2 had the most stable expression in different flower developmental stages while YLS8, HIS3.3 and ACT7 were the top-ranking reference genes for normalisation in mutant studies. Furthermore, we validated our results by analysing the expression pattern of genes involved in reproduction and examining the expression of these genes in published mutant backgrounds. Overall, we provided a pool of appropriate reference genes for expression studies in reproductive tissues of A. thaliana, which will facilitate further gene expression studies in this context. More importantly, we presented a framework that will promote a consistent and accurate analysis of gene expression in any scientific field. Simultaneously, we highlighted the relevance of clearly defining and describing the experimental conditions associated with qPCR to improve scientific reproducibility.

Reference Genes Screening and Gene Expression Patterns Analysis Involved in Gelsenicine Biosynthesis under Different Hormone Treatments in Gelsemium elegans

“…RT-qPCR is one of the most accurate technique to analyze gene expression profiles, and the screening of optimal reference genes is a crucial step [45]. In many plant species, optimal reference genes have been identified under various experimental conditions [46]. At present, EF-1α and TUA are the best two genes for Chenopodium quinoa seedlings after treating with ABA [47].…”

Identification of MiR398 and Its Regulatory Roles in Terpenoid Biosynthesis of Persicaria odorata

“…It is important to minimise the variability due to sample preparation, RNA quantity, and PCR efficiency [40]. Hence, reference gene shown to aid minimising variations by normalising the target gene's mRNA level or target miRNA level with the reference gene's mRNA level after reverse transcription the sequences into cDNA [29,41,42].…”

Advancements in Reference Gene Selection for Fruit Trees: A Comprehensive Review

“…They exhibit ubiquitous expression across diverse cell types, underscoring their indispensable roles in fundamental biological processes. In investigations focusing on gene expression analyses in various plant species such as Arabidopsis thaliana, Oryza sativa, and Zea mays, the genes actin (ACT), α-tubulin (α-Tua), β-tubulin (β-Tub), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and elongation factor-1α (EF-1α), among others, have gained prominence as frequently employed internal reference genes [26][27][28][29]. Their consistent usage as reference points facilitates accurate normalization and reliable quantification of gene expression levels in a wide range of plant-based research endeavors (Table 1).…”