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Abstract
Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique to quantify gene expression. To facilitate gene expression study and obtain accurate results, normalization relative to stably expressed reference genes is crucial. The monarch butterfly, Danaus plexippus (L.), is one of the most recognized insect species for its spectacular annual migration across North America. Besides its great voyages, D. plexippus has drawn attention to its role as a bio-indicator, ranging from genetically modified organisms (GMOs) to natural ecosystems. In this study, nine reference genes from D. plexippus genome were selected as the candidate reference genes. The expression profiles of these candidates under various biotic and abiotic conditions were evaluated using the four readily available computational programs, BestKeeper, Normfinder, geNorm, and ΔCt method, respectively. Moreover, RefFinder, a web-based computational platform integrating the four above mentioned algorisms, provided a comprehensive ranking of the stability of these reference genes. As a result, a suite of reference genes were recommended for each experimental condition. Specifically, elongation factor 1α (EF1A) and ribosomal protein 49 (RP49) were the most stable reference genes, respectively, under biotic (development, tissue, and sex) and abiotic (photoperiod, temperature, and dietary RNAi) conditions. With the recent release of a 273-million base pair draft genome, results from this study allow us to establish a standardized RT-qPCR analysis and lay a foundation for the subsequent genomic and functional genomic research in D. plexippus, a major bio-indicator and an emerging model for migratory animals.
Highlights
The advent of next-generation sequencing technologies has led to a significant increase in transcriptomic and genomic output for various organisms [1,2,3]
The monarch butterfly is commonly used as a non-target insect for the ecological risk assessment of transgenic crops in the U.S.; plus interest in the migratory biology and genetics of this insect suggest future post-genome studies will occur
Expression profiles of nine candidate reference genes from D. plexippus were evaluated under diverse experimental conditions
Summary
The advent of next-generation sequencing technologies has led to a significant increase in transcriptomic and genomic output for various organisms [1,2,3]. In RTqPCR, a commonly used technique to normalize the gene expression data is to introduce one or multiple reference genes, which are stably expressed across various experimental conditions and serve as the internal control [6, 9, 10]. These reference genes have been defined functionally as ‘constitutively expressed to maintain cellular function’, they do not necessarily meet prerequisites for a good reference gene that can be ‘expressed at constant levels across various biotic and abiotic conditions [4, 8, 10]. Many studies have proved that some commonly used reference genes express differentially across different experimental conditions; researchers have documented that multiple reference genes should be used for accurate normalization [4, 8, 9, 11, 12]
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