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Title: Selection of reference genes for RT-qPCR analysis in the monarch butterfly, Danaus plexippus (L.), a migrating bio-indicator

Author
item PAN, HUIPENG - University Of Kentucky
item TANG, XIAOWEI - University Of Kentucky
item Bidne, Keith
item Hellmich Ii, Richard
item SIEGFRIED, BLAIR - University Of Nebraska
item ZHOU, XUGUO - University Of Kentucky

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/9/2015
Publication Date: 6/1/2015
Citation: Pan, H., Tang, X., Bidne, K.G., Hellmich II, R.L., Siegfried, B., Zhou, X. 2015. Selection of reference genes for RT-qPCR analysis in the monarch butterfly, Danaus plexippus (L.), a migrating bio-indicator. PLoS One. 10(6):e0129482. DOI: 10.1371/journal.pone.0129482.

Interpretive Summary: All organisms express different combinations of genes at various times of development and under different environmental conditions. Scientists use quantitative real-time polymerase chain reaction (qRT-PCR) to measure and evaluate such changes in gene expression. To facilitate these gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. In this study, nine candidate reference genes were evaluated for the monarch butterfly, Danaus plexippus. These genes included: elongation factor 1a (EF1A), glyceralde hyde-3-phosphate dehydro-genase (GAPDH), nicotinamide adenine dinucleotide (NADH), cyclophilins A (CypA), ribosomal protein S5 (RPS5), ribosomal protein 49 (RP49), vacuolar-type H+-ATPase (v-ATPase), 28S ribosomal RNA (28S), and 18S ribosomal RNA (18S). Based on the results, EF1A was the most suitable gene for developmental time, tissue, and gender characteristics; RP49 was the most suitable gene for photoperiod, temperature, and double-stranded RNA feeding. A suite of candidate reference genes were recommended for selected experimental conditions. This work sheds light on establishing a standardized qRT-PCR procedure for the monarch butterfly and serves as a starting point for screening for reference genes for expression studies of related insects. This work will be useful to all scientists interested in insect gene expression, especially butterfly gene expression.

Technical Abstract: Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring and evaluating changes in gene expression. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. In this study, expression profiles of nine candidate reference genes including elongation factor 1a (EF1A), glyceralde hyde-3-phosphate dehydro-genase (GAPDH), nicotinamide adenine dinucleotide (NADH), cyclophilins A (CypA), ribosomal protein S5 (RPS5), ribosomal protein 49 (RP49), vacuolar-type H+-ATPase (v-ATPase), 28S ribosomal RNA (28S), and 18S ribosomal RNA (18S) from the monarch butterfly, Danaus plexippus (L.), and four alternative methods (geNorm, Normfinder, BestKeeper, and 'Ct method) were used to evaluate the suitability of these genes as endogenous controls under diverse experimental conditions. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, EF1A was the most suitable gene under biotic factors (developmental time, tissue, and sex), RP49 was the most suitable gene under abiotic factors (photoperiod, temperature, and dsRNA feeding). Nevertheless, a suite of candidate reference genes were specifically recommended for selected experimental conditions. This work sheds light on establishing a standardized qRT-PCR procedure in the migration and eco-indicator insect D. plexippus, and serves as a starting point for screening for reference genes for expression studies of related insects.