RRC ID 81249
Author Ferreira MJ, Silva J, Pinto SC, Coimbra S.
Title I Choose You: Selecting Accurate Reference Genes for qPCR Expression Analysis in Reproductive Tissues in Arabidopsis thaliana.
Journal Biomolecules
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.
Volume 13(3)
Published 2023-3-2
DOI 10.3390/biom13030463
PII biom13030463
PMID 36979397
PMC PMC10046263
MeSH Arabidopsis* / genetics Flowers / genetics Gene Expression Profiling Gene Expression Regulation, Plant Real-Time Polymerase Chain Reaction / methods Reproducibility of Results
Resource
Arabidopsis / Cultured plant cells, genes pst05619 pst11704