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

Jun-Yi Li,Xin Huang,Chun Chen,Wan-Zhu Chen,Hui Xie,Chun-Ling Xu,Si-Hua Yang

doi:10.7717/peerj.6253

Jun-Yi Li, Xin Huang + Show 5 more

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https://doi.org/10.7717/peerj.6253

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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.

Highlights

Polymerase chain reaction (PCR) is a technique that can amplify certain DNA fragments in vitro (Saiki et al, 1985)
The PCR amplification fragments of candidate reference genes actin, Rps21, eIF5A, atubulin, UBI and β-PP1 from R. similis are 382 bp, 344 bp, 556 bp, 538 bp, 473 bp and 719 bp, respectively (Fig. 1), and all are consistent with the expected size and the corresponding sequence of these genes from the transcriptome of R. similis
The sequences of the fragments mentioned above were used to perform a BLAST search of the NCBI nonredundant protein database; the results showed that the amino acid sequences are highly similar between the six candidate reference gene fragments and the corresponding genes of other nematodes (Table 5)

Summary

Introduction

Polymerase chain reaction (PCR) is a technique that can amplify certain DNA fragments in vitro (Saiki et al, 1985). Real-time quantitative polymerase chain reaction (Real-time PCR), a modified PCR technique that adds a fluorescent dye or fluorescent probe to the reaction system during the PCR reaction, enables quantitative analysis of an initial template that is positively correlated with its product by real-time monitoring of fluorescence signal strength changes, achieving real-time detection of each round of PCR reaction products (Ginzinger & David, 2002; Schmittgen, 2001). Compared with end-point quantitation of ordinary PCR techniques, real-time-PCR is superior in terms of accuracy, repeatability, specificity, sensitivity and convenient operation; it has been widely used in various fields of molecular biology (Bustin & Dorudi, 1998; Gachon, Mingam & Charrier, 2004; Higuchi et al, 1993). Screening of reference genes in real-time PCR for Radopholus similis.

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