Reference gene stability of a synanthropic fly, Chrysomya megacephala

Abstract

BackgroundStable reference genes are essential for accurate normalization in gene expression studies with reverse transcription quantitative polymerase chain reaction (qPCR). A synanthropic fly, Chrysomya megacephala, is a well known medical vector and forensic indicator. Unfortunately, previous studies did not look at the stability of reference genes used in C. megacephala.ResultsIn this study, the expression level of Actin, ribosomal protein L8 (Rpl8), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1), α-tubulin (α-TUB), β-tubulin (β-TUB), TATA binding box (TBP), 18S rRNA (18S) and ribosomal protein S7 (Rps7) were evaluated for their stability using online software RefFinder, which combines the normal software of the ΔCt method, BestKeeper, Normfinder, and geNorm. Moreover the number of suitable reference gene pairs was also suggested by Excel-based geNorm. The expression levels of these reference genes were evaluated under different experimental conditions with special perspectives of forensic applications: developmental stages (eggs, first, second and third instar larvae, pupae and adults); food sources of larvae (pork, fish and chicken); feeding larvae with drugs (untreated control, Estazolam and Marvelon); feeding larvae with heavy metals (untreated control, cadmium and zinc); tissues of adults (head, thorax, abdomen, legs and wings). According to RefFinder, EF1 was the most suitable reference gene of developmental stages, food and tissues; 18S and GAPDH were the most suitable reference genes for drugs and heavy metals, respectively, which could be widely used for quantification of target gene expression with qPCR in C. megacephala. Suitable reference gene pairs were also suggested by geNorm.ConclusionThis fundamental but vital work should facilitate the gene studies of related biological processes and deepen the understanding in physiology, toxicology, and especially medical and forensic entomology of C. megacephala.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1175-9) contains supplementary material, which is available to authorized users.

Reference gene selection for quantitative gene expression analysis in black soldier fly (Hermetia illucens)

“…Therefore, normalization is the key to achieve accurate target gene results [20, 21]. Traditionally, housekeeping genes such as beta-actin ( Actin ), translation elongation factor 1-alpha ( EF1 ), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), alpha-tubulin ( Tubulin ) and TATA - binding box ( TBP ) have been used as reference genes for the normalization of qPCR data [22, 23]. However, the expression of these reference genes fluctuates substantially under different experimental conditions [24].…”

Selection of reference genes for quantitative real‐time polymerase chain reaction normalization inBradysia odoriphaga(Diptera: Sciaridae)

“…) and Chrysomya megacephala (Wang et al . ). In this study, RPS15 was the second optimal reference gene across different developmental stages in B. odoriphaga , and 18S , RPS15 and RPS7 were considered suitable reference genes across developmental stages.…”

Evaluation of reference genes and expression of key genes involved in the isoprenoid metabolic pathway of rice leaves after infection by the Southern rice black-streaked dwarf virus

“…The stability of expression of each candidate reference gene was ranked using three different types of Microsoft Excel-based software: geNorm [29], Normfinder [30] and BestKeeper [31]. In addition, RefFinder [32] was used to rank the overall stability of all candidate reference genes. The relative expression levels of the key genes involved in the isoprenoid metabolic pathway in rice leaves infected by SRBSDV were quantified using the 2 −ΔΔCt method [33].…”