# Study on the Stability of Reference Genes and HSP60 for Expression Analysis in Chilo suppressalis in Response to Humidity Stress

## TL;DR

This study identifies the most stable reference genes for qRT-PCR in the insect Chilo suppressalis under humidity stress, improving gene expression analysis.

## Contribution

The study provides validated reference genes for different developmental stages of C. suppressalis under humidity stress.

## Key findings

- 18S rRNA was the most stable reference gene in third instar larvae.
- Using two reference genes improved data normalization across all tested conditions.
- Hsp60 expression varied significantly depending on the reference gene used for normalization.

## Abstract

Quantitative real-time PCR (qRT-PCR) is necessary to select stable reference genes for normalization. In this study, the suitability of various reference genes for qRT-PCR analysis was evaluated in different developmental stages of Chilo suppressalis exposed to desiccation or rehydration stress. The results indicated that 18S rRNA was the most stable reference gene for monitoring gene expression in the third instar larvae; ACTIN, TUB, UBI, UBI, and EF1 were the optimal genes for the fifth instar larvae, male pupae, female pupae, male adults, and female adults, respectively. The optimal number of reference genes recommended by geNorm analysis indicated that two candidate reference genes were sufficient for data normalization under all experimental conditions tested. To validate these recommendations, the expression profile of Hsp60 was investigated. Hsp60 transcript levels showed significant differences when normalized to the most stable single reference gene, or combined reference genes, compared with the least stable reference gene. The reference genes identified in the present study will enhance the reliability of gene expression data for C. suppressalis under humidity stress.

Quantitative real-time PCR (qRT-PCR) is a high-reliability, -sensitivity, and -operability technique to quantify gene expression. It is necessary to select stable reference genes for normalization. Water plays important roles in the metabolism, physiology, distribution, and so on, in insects. In this study, the suitability of various reference genes for qRT-PCR analysis was evaluated in different developmental stages of Chilo suppressalis exposed to desiccation or rehydration stress. The ∆Ct method, geNorm, NormFinder, and BestKeeper were used to evaluate the suitability of nine reference genes for normalizing gene expression in the third instar larvae, the fifth instar larvae, male pupae, female pupae, male adults, and female adults under different humidities. The results indicated that 18S rRNA was the most stable reference gene for monitoring gene expression in the third instar larvae, while ACTIN, TUB, UBI, UBI, and EF1 were the optimal genes for the fifth instar larvae, male pupae, female pupae, male adults, and female adults, respectively. The optimal number of reference genes recommended by geNorm analysis indicated that two candidate reference genes were sufficient for data normalization under all experimental conditions tested. To validate these recommendations, the expression profile of the gene encoding heat shock protein 60 (Hsp60) was investigated. Hsp60 transcript levels showed significant differences when normalized to the most stable single reference gene, or combined reference genes, compared with the least stable reference gene. The reference genes identified in the present study will enhance the reliability of gene expression data for C. suppressalis under humidity stress.