# Validation of Stable Reference Genes for RT-qPCR Normalization in Oxycetonia jucunda (Coleoptera: Scarabaeidae)

## TL;DR

This study identifies stable reference genes for RT-qPCR in Oxycetonia jucunda, a pest that damages fruit trees, to improve gene expression analysis.

## Contribution

The study provides the first systematic evaluation of reference genes for RT-qPCR normalization in O. jucunda across multiple tissues.

## Key findings

- RPS3 and RPS31 were identified as the most stably expressed reference genes across six adult tissues of O. jucunda.
- The stability of seven candidate reference genes was evaluated using five computational methods.
- The selected reference genes were validated by normalizing the expression of the OBP3 target gene.

## Abstract

Oxycetonia jucunda Faldermann is a polyphagous pest that inflicts damage on a range of fruit tree species. The selection of an appropriate reference gene is essential for the reliable analysis of gene expression when using real-time quantitative polymerase chain reactions. This study aimed to identify stable internal controls by evaluating the stability of seven candidate reference genes across several O. jucunda tissues and assessing their suitability using five different algorithms. To verify the screening results, we examined the expression patterns of the odor-binding protein gene OBP3. The most stable reference genes identified using these analyses will provide a basis for further molecular studies on O. jucunda.

The polyphagous pest Oxycetonia jucunda Faldermann can cause substantial damage to a range of economically important crops, with the adult beetles feeding directly on floral tissues and young leaves. RT-qPCR is widely used to analyze gene expression, for which the selection of stable reference genes is essential for enabling an accurate normalization of expression. However, no systematic evaluations of suitable reference genes for RT-qPCR analysis using different tissues of O. jucunda have been conducted. To assess their applicability as reliable normalization controls, we used five computational methods to examine the stability of seven potential reference genes (GAPDH, EF1α, RPS3, RPS18, RPL18, RPS31, and UBC5A) across six adult tissues, with three biological replicates per tissue. The findings revealed RPS3 and RPS31 to be the most stably expressed. This pair of reference genes was further validated by normalizing the expression of the odorant-binding protein 3 (OBP3) target gene. Our findings will provide important foundational data for the accurate analysis of functional gene expression in O. jucunda.