Abstract
To accurately evaluate gene expression levels and obtain more accurate quantitative real-time RT-PCR (qRT-PCR) data, normalization relative to reliable reference gene(s) is required. Drosophila suzukii, is an invasive fruit pest native to East Asia, and recently invaded Europe and North America, the stability of its reference genes have not been previously investigated. In this study, ten candidate reference genes (RPL18, RPS3, AK, EF-1β, TBP, NADH, HSP22, GAPDH, Actin, α-Tubulin), were evaluated for their suitability as normalization genes under different biotic (developmental stage, tissue and population), and abiotic (photoperiod, temperature) conditions. The three statistical approaches (geNorm, NormFinder and BestKeeper) and one web-based comprehensive tool (RefFinder) were used to normalize analysis of the ten candidate reference genes identified α-Tubulin, TBP and AK as the most stable candidates, while HSP22 and Actin showed the lowest expression stability. We used three most stable genes (α-Tubulin, TBP and AK) and one unstably expressed gene to analyze the expression of P-glycoprotein in abamectin-resistant and sensitive strains, and the results were similar to reference genes α-Tubulin, TBP and AK, which show good stability, while the result of HSP22 has a certain bias. The three validated reference genes can be widely used for quantification of target gene expression with qRT-PCR technology in D.suzukii.
Highlights
Quantitative real-time PCR is a fast, sensitive, repeatable and accurate method for quantifying gene transcript levels [1,2]
In order to select the most suitable reference gene(s) for gene expression quantification by Quantitative real-time PCR (qRT-PCR), we examined the expressions of ten candidate reference genes (RPL18, ribosomal protein S3 (RPS3), arginine kinase (AK), elongation factor 1 beta (EF-1b), TATA binding protein (TBP), NADH, heat shock protein 22 (HSP22), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Actin, a-Tubulin) in four developmental stages, three body regions, three different populations, and two abiotic conditions treated samples
The primer efficiencies of each standard curve for PCR efficiency, which was generated from third-instar larvae of the laboratory strain, were ranging from the lowest for ribosomal protein L18 (RPL18) (92.6%) to the highest for AK (109.4%)
Summary
Quantitative real-time PCR (qRT-PCR) is a fast, sensitive, repeatable and accurate method for quantifying gene transcript levels [1,2]. Several studies have revealed that interpretation would produce appreciable errors owing to different samples, treatments, RNA extraction techniques, amplification efficiencies, etc. It requires an appropriate candidate reference gene to normalize the target gene expression, and the reference genes must have stable expression existing features in different cell types and experimental conditions [3,4,5]. There are few research reports on D. suzukii, and so far no qRT-PCR studies on it have been reported, so there is especially important to validated reference gene(s) for it
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