Selection and Validation of Reference Genes for RT-qPCR Analysis of Gene Expression in Nicotiana benthamiana upon Single Infections by 11 Positive-Sense Single-Stranded RNA Viruses from Four Genera

Abstract

Quantitative real-time PCR (RT-qPCR) is a widely used method for studying alterations in gene expression upon infections caused by diverse pathogens such as viruses. Positive-sense single-stranded (ss(+)) RNA viruses form a major part of all known plant viruses, and some of them are damaging pathogens of agriculturally important crops. Analysis of gene expression following infection by ss(+) RNA viruses is crucial for the identification of potential anti-viral factors. However, viral infections are known to globally affect gene expression and therefore selection and validation of reference genes for RT-qPCR is particularly important. In this study, the expression of commonly used reference genes for RT-qPCR was studied in Nicotiana benthamiana following single infection by 11 ss(+) RNA viruses, including five tobamoviruses, four potyviruses, one potexvirus and one polerovirus. Stability of gene expression was analyzed in parallel by four commonly used algorithms: geNorm, NormFinder, BestKeeper, and Delta CT, and RefFinder was finally used to summarize all the data. The most stably expressed reference genes differed significantly among the viruses, even when those viruses were from the same genus. Our study highlights the importance of the selection and validation of reference genes upon different viral infections.

Interplay between a polerovirus and a closterovirus decreases aphid transmission of the polerovirus

“…To select reference genes, we used a list of stably expressed plant genes previously established on Arabidopsis thaliana infected by several viruses (Zhang et al, 2023) . Orthologous genes were identified in Beta vulgaris using the KEGG database and primers compatible with TaqMan and SYBR Green detection were designed using the IDT Primer Request tool available online (https://eu.idtdna.com/pages/tools/primerquest).…”

Unveiling the Role of SlRNC1 in Chloroplast Development and Global Gene Regulation in Tomato Plants

“…Amplification of the genomes was achieved using KOD FX Neo high-fidelity DNA polymerase (TOYOBO, Japan) with specific primers. For quantitative PCR (qPCR), AceQ qPCR SYBR Green PCR Master Mix kit (Vazyme, Nanjing, China) was utilized, with the specificity of each reaction confirmed through melting curve analysis [42]. RT-qPCR was carried out on a LightCycler480 instrument (Roche, Basel, Switzerland) with 384-well PCR plates.…”

“…Three biological replicates were included for each experiment, and three technical replicates were set for each biological replicate. The thermal cycling conditions for RT-qPCR were 95 • C for 30 s, followed by 40 cycles of 15 s at 95 • C, 14 s at 55 • C, and 28 s at 72 • C. Melting curves were prepared with the following thermal conditions: 95 • C for 10 s, 60 • C for 30 s, and 95 • C for 15 s. Relative gene expression was analyzed using the 2 −∆∆CT method [42]. All primers utilized in this study are listed in Table S1.…”

Differential Participation of Plant Ribosomal Proteins from the Small Ribosomal Subunit in Protein Translation under Stress

“…The RT-qPCR was performed using the CFX Connect detection system (Bio-Rad Laboratories, Mississauga, ON, Canada). ACT 1 and EF1α were used to normalize the transcript levels [51]. Each sample was run in triplicate and repeated six times from two pooled biological replicates of silenced and non-silenced plants.…”