Selection of reference genes for expression analysis of plant-derived microRNAs in Plutella xylostella using qRT-PCR and ddPCR

The establishment of an expression quantification system that can be easily applied for the comparison of microRNAs (miRNAs) from biological samples is an important step toward understanding functional mechanisms in organisms. However, there is lack of attention on the selection of reference genes for miRNA expression profiling in insect herbivores. Here, we explored the candidate reference genes in a notorious pest of cruciferous crops, Plutella xylostella, for normalization of miRNA expression in developmental stages and tissues and in response to a change of food source from artificial diet to host plant Arabidopsis thaliana. We first compared the expression levels and stability of eight small RNAs using qRT-PCR, and found that miR11 was the most suitable reference gene for expression quantification of the miRNAs. We then confirmed this finding using digital droplet PCR and further validated with a well-studied cross-kingdom miRNA derived from A. thaliana (ath-miR159a). However, none of the reference genes was applicable for all experimental conditions, and multiple reference genes were sometimes required within the same experiment. Our work provides a method for the selection of reference genes for quantification of plant-derived miRNAs, which paves the way for unveiling their roles in the insect-plant coevolution.