Validation of reference genes for qRT-PCR analysis in Megoura viciae (Hemiptera Aphididae)

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) has become one of the most sensitive methods to monitor gene expression. An important and often neglected requirement of this kind of study is the validation of appropriate reference genes with the most possible stable expression levels across samples groups. In this paper, several candidates were tested in all the four nymphal instars and the adult morphs (winged and apterous) of aphid Megoura viciae Buckton (Hemiptera Aphididae), an important pest of broad bean, in order to obtain reference genes for future works on Megoura viciae gene expression. Since the use of multiple reference genes is recommended for an accurate normalization, eight candidate genes were tested, encoding respectively for: ribosomal protein L32 (RPL32), NADH dehydrogenase (ubiquinone) flavoprotein 1 (NADH), succinate dehydrogenase complex subunit A (SUCC), ribosomal protein S9 (RPS9), TATA-box binding protein (TATA), actin (ACT), β-tubulin (TBU) and ubiquitin-conjugating protein (UBIQ). Three software programs and the comparative ΔCT method were used to compare and rank the candidate genes and RefFinder - a web-based comprehensive tool that integrates all the four methods in a single index - indicated RPS9-RPL32 as the best couple. In addition, our study showed that a common-used reference gene, β-actin, achieved the worst score among our candidates. Finally, differences between the “classic” normalization with β-actin as a reference gene and the normalization using the best reference genes according to our work were highlighted using for the first time a Megoura viciae odorant binding protein, OBP4, as the target gene.