Evaluation of reference genes for real-time PCR quantification of gene expression in the Australian sheep blowfly, Lucilia cuprina.
The Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae), is an important pest of sheep in Australia and other parts of the world. However, the paucity of information on many fundamental molecular aspects of this species limits the ability to exploit functional genomics techniques for the discovery of new drug targets for its control. The present study aimed to facilitate gene expression studies in this species by identifying the most suitable reference genes for normalization of mRNA expression data. Quantitative real-time polymerase chain reaction (PCR) was performed with 11 genes across RNA samples from eggs, L1, L3, pupae and adult life stages, and two normalization programs, Normfinder and geNorm, were then applied to the data. The results showed an ideal set of genes (18S rRNA, 28S rRNA, GST1, beta-tubulin and RPLPO) for data normalization across all life stages. The most suitable reference genes for studies within specific life stages were also identified. GAPDH was shown to be a poor reference gene. The reference gene recommendations in this study will be of use to laboratories investigating gene expression in L. cuprina and related blowfly species.
Bagnall
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CSIRO (Commonwealth Scientific & Industrial Research Organization) Livestock Industries, St. Lucia, Brisbane, Queensland 4067, Australia.
Kotze
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D000818:Animals; D004175:Diptera / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation; D017344:Genes, Insect / Q000235:genetics*; D008018:Life Cycle Stages / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction