Selection and validation of reference genes for quantitative expression analysis of regeneration-related genes in Cheilomenes sexmaculata by real-time qRT-PCR.

Abstract

Regeneration is a fascinating phenomenon that has intrigued scientists for a long time. Cheilomenes sexmaculata (Fabricius), a zig-zag ladybird beetle, possesses a high capacity for limb regeneration. The molecular mechanics of the zig-zag ladybird beetle are under-explored. Current research trends are focused on uncovering functional genes associated with limb regeneration. Most of these investigations involve quantitative real-time PCR (qRT-PCR) for their rapid and accurate analysis of gene expression levels. Hence, a stable and suitable reference gene is required to normalize the gene expression data. In this study, five housekeeping genes were selected from the transcriptomics data (in-house unpublished data) of C. sexmaculata (Fabricius). The expression stabilities of the selected genes were evaluated under different time intervals post-amputation using geNorm, normFinder, and refFinder software. Actin was revealed to be the most stable housekeeping gene, along with elongation factor 2 and glyceraldehyde-3-phosphate dehydrogenase. A target gene named engrailed (an important segment-forming gene) was used to validate the selected reference genes. The expression levels were found to be consistent with the transcriptomics results. According to our study, actin, along with elongation factor 2 and glyceraldehyde-3-phosphate dehydrogenase, serve as the most stable reference genes and are suitable for regeneration-related research. This study is a groundbreaking effort to identify the most stable reference gene for limb regeneration in C. sexmaculata (Fabricius), and the findings can be applied to other related insect species.