Evaluation and validation of experimental condition-specific reference genes for normalization of gene expression in...

Functional genomics in whitefly, Bemisia tabaci is gaining impetus due to its polyphagous nature, worldwide distribution and recently sequenced whole genome. These studies require an in-depth evaluation and validation of reference genes in different development stages and variable experimental setups. Normalization with reference genes is an essential step in the gene expression studies. Rather than selecting a reference gene empirically, the suitability of these genes must be validated for an individual organism, its specific stage or even for particular experimental conditions. The Quantitative real-time polymerase chain reaction (RT-qPCR) has evolved as an efficient and widely used technique for precise monitoring of gene expression. The prime focus of this study was to identify candidate reference genes in different developmental stages (adults, nymphs, eggs), sex (male and female), hosts (Gossypium hirsutum, G. arboreum), and under insecticidal and starvation stress. Expression stability of these genes in different experimental samples was evaluated by employing five different computational algorithms such as NormFinder, BestKeeper, Comparative delta-CT, geNorm and RefFinder. Our results identified a different set of reference genes under each experimental setup such as electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), ubiquitin (UBIQ) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as best reference genes in adult whitefly. In addition, glutathione S-transferase (GST) in eggs, cyclophilin (CYCLOPH) in red eyed nymph, GAPDH in third instar, tubulin (Tub) in female, ubiquitin ribosomal protein S27 (UBIRPS2) in male, succinate dehydrogenase complex subunit B (SDHB) under insecticidal stress, ETF-QO under starvation stress, UBIRPS2 under host influence were the top most stable genes. Our studies report the importance of selection of specific reference genes for accurate gene expression studies under various experimental setups in B. tabaci.