Identification and validation of reference genes for quantitative real-time PCR in Drosophila suzukii (Diptera: Drosophilidae)

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...

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Bibliographic Details
Published in:PloS one 2014-09, Vol.9 (9), p.e106800-e106800
Main Authors: Zhai, Yifan, Lin, Qingcai, Zhou, Xianhong, Zhang, Xiaoyan, Liu, Tingli, Yu, Yi
Format: Article
Language:English
Subjects:
Abamectin
Actin
Adults
Animals
Base Sequence
Biology and Life Sciences
Dehydrogenases
Diptera
DNA Primers
Drosophila
Drosophila - genetics
Drosophila suzukii
Drosophilidae
Eggs
Fruits
Gene expression
Genes
Glyceraldehyde-3-phosphate dehydrogenase
Glycoproteins
Hemiptera
Insecticides
Insects
Kinases
Laboratories
Medical research
NADH
Nicotinamide adenine dinucleotide
P-Glycoprotein
Polymerase chain reaction
Population (statistical)
Proteins
Real time
Real-Time Polymerase Chain Reaction
Stability
Tubulin
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0106800