Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress

TLDR

Gene expression studies increasingly rely on real‑time RT‑PCR, the most sensitive method for detecting low‑abundance mRNA, which demands precise and reproducible measurements. The study aimed to evaluate the stability of seven candidate housekeeping genes in potato under biotic (late blight) and abiotic (cold, salt) stresses using geNorm. Real‑time RT‑PCR was performed with the seven housekeeping genes as internal controls, and geNorm software assessed their expression stability, while also examining hsp20.2 variability to illustrate the impact of control choice. ef1α proved the most stable reference gene, whereas the others fluctuated with stress, and hsp20.2 quantification varied depending on the internal control(s) used, underscoring the critical role of selecting appropriate controls.

Abstract

Plant stress studies are more and more based on gene expression. The analysis of gene expression requires sensitive, precise, and reproducible measurements for specific mRNA sequences. Real-time RT-PCR is at present the most sensitive method for the detection of low abundance mRNA. To avoid bias, real-time RT-PCR is referred to one or several internal control genes, which should not fluctuate during treatments. Here, the non-regulation of seven housekeeping genes (beta-tubulin, cyclophilin, actin, elongation factor 1-alpha (ef1alpha), 18S rRNA, adenine phosphoribosyl transferase (aprt), and cytoplasmic ribosomal protein L2) during biotic (late blight) and abiotic stresses (cold and salt stress) was tested on potato plants using geNorm software. Results from the three experimental conditions indicated that ef1alpha was the most stable among the seven tested. The expression of the other housekeeping genes tested varied upon stress. In parallel, a study of the variability of expression of hsp20.2, shown to be implicated in late blight stress, was realized. The relative quantification of the hsp20.2 gene varied according to the internal control and the number of internal controls used, thus highlighting the importance of the choice of internal controls in such experiments.