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Selection and validation of reference genes for quantitative expression analysis of regeneration-related genes in
Shivali Pandita1,2, Hera Alam2, Radha Shivhare3
1Molecular Biology and Biotechnology Division, CSIR-National Botanical Research Institute, (Council of Scientific and Industrial Research) Rana Pratap Marg, Lucknow, UP, 226001, India.
Background:
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.
Methods And Results:
In this study, five housekeeping genes were selected from the transcriptomics data (in-house unpublished data) of C. sexmaculata (Fabricius).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.
Conclusion:
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.
