Screening of reference genes in real-time PCR for Radopholus similis

Abstract

Six candidate reference genes were chosen from the transcriptome database of Radopholus similis using the bioinformatics method, including four conventional reference genes (actin, Eukaryotic translation initiation factor 5A (eIF5A), Tubulin alpha (a-tubulin), ubiquitin (UBI)) and two new candidate reference genes (Ribosomal protein S21 (Rps21) and Serine/threonine protein phosphatase PP1-β catalytic subunit (β-PP1)). In addition, a traditional reference gene 18S ribosomal RNA (18S rRNA) obtained from NCBI databases was also added to the analysis. Real-time PCR was used to detect the expression of seven candidate reference genes in six populations of R. similis and four developmental stages (female, male, larva and egg) of a population. The stability of the expression of candidate genes was evaluated by three software programs, BestKeeper, geNorm and NormFinder. The results showed that eIF5A is the most suitable reference gene for gene functional research of different populations, while both Rps21 and eIF5A are the most suitable reference genes for different developmental stages of a population. Therefore, eIF5A is the best reference gene for studying R. similis. However, one defect of this study is that only seven candidate reference genes were analyzed; ideally, more genes should be tested.

A Venom Allergen-Like Protein, RsVAP, the First Discovered Effector Protein of Radopholus similis That Inhibits Plant Defense and Facilitates Parasitism

“…Total RNA was extracted from the nematodes using the RNAprep pure micro kit (Tiangen, Beijing, China) following the manufacturer’s instructions; cDNA was synthesized using performing a reverse transcription of the extracted RNA using one-step gDNA removal and cDNA synthesis SuperMix (TransGen, Beijing, China). One specific primer pair, QVAP-F/QVAP-R ( Supplementary Table S1 ), was designed for quantitative reverse-transcription PCR (RT-qPCR) according to the full-length mRNA sequence of RsVAP , and eif5a [ 57 ] was used as an internal reference gene. RT-qPCR was performed using AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China) on the Bio-Rad CFX-96 system (Bio-Rad Laboratories, Hercules, CA, USA).…”

Identification and validation of reference genes for real-time RT-PCR in Aphelenchoides besseyi

“…Eleven candidate reference genes were chosen from RNA-seq data of Globodera rostochiensis to identify a reliable set of reference genes to study gene expression, three genes including GR, PMP-3 and aaRS were found to be stable in different stages of G.rostochiensis (Sabeh et al [9]). eIF5a were identified as a suitable reference gene that could be expressed stably in different developmental stages and different populations of Radopholus similis [10]. Such studies confirmed that not all traditional reference genes are suitable for a qPCR relative quantitative analysis under all experimental conditions; the most suitable reference gene may change as the experimental conditions change.…”

“…In another paper published by our group, eIF5A was tested as the best reference genes for R. similis [10]. Both papers analyzed the expression stability of 18 s rRNA, actin, and α-tubilin in two nematode species.…”

Targeted transcriptomics reveals signatures of large-scale independent origins and concerted regulation of effector genes in Radopholus similis

“…The same set of primers selected for in situ hybridization were used for nematode transcript amplification. The following genes were used as references: the αtubulin gene for R. similis [84], while the translation elongation factor EF-1 alpha gene (EF-1α) of carrot was used as host reference gene [85]. Two independent RNA-seq libraries were generated from a pool of nematode stages collected from carrot roots (30 DAI).…”