Evaluation of reference genes expression stability by quantitative real-time PCR in Tribolium castaneum larvae after bacterial infection

Quantitative real-time PCR (qRT-PCR) is a commonly used technique for analyzing gene expression levels. The selection of a relatively stable reference gene under experimental conditions is crucial for data normalization.

This study aimed to evaluate the expression stability of candidate reference genes in Tribolium castaneum larvae under different bacterial infections, laying a foundation for subsequent gene expression analysis in T. castaneum.

Larvae of T. castaneum were infected with Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus by feeding. The relative expression levels of eight candidate reference genes, including ACTB, CAD, RpL13A, RpL32, RpS3, RpS6, RpS18, and Syx1A, were detected by qRT-PCR across different tissues and at various infection time points. The expression stability of these candidate reference genes was comprehensively evaluated using ΔCq, geNorm, NormFinder, and BestKeeper.

Based on qRT-PCR results, RpS3 and RpL32 were identified as the most stable reference genes in the non-infected control group. During E. coli infection, RpS3 and RpL32 remained the most stable genes, while Syx1A and ACTB showed relatively high stability. For S. aureus infection, RpS3, RpL32, and Syx1A were the most stable genes. Comprehensive analysis revealed that RpS3 and RpL32 were the two most stably expressed genes across different tissues and after both bacterial infections, while CAD exhibited the poorest expression stability.

Therefore, RpS3 and RpL32 are recommended as reference genes for qRT-PCR analysis in the larvae of T. castaneum after bacterial infection. Syx1A and ACTB can also be used as the candidate reference genes.