Identification of stable reference genes for quantitative PCR in koalas.

Area of Science:

• Veterinary Immunology
• Molecular Biology
• Genomics

Background:

• Accurate gene expression studies are vital for understanding host and immune responses to diseases.
• Reference genes with stable expression are essential for normalizing gene expression data in such studies.

Purpose of the Study:

• To identify and validate stable reference genes in koala lymph node tissues.
• To compare gene expression stability across two genetically distinct koala populations.

Main Methods:

• Transcriptome analysis to identify candidate stable genes.
• Verification of 11 candidate genes and two commonly used genes (ACTB, GAPDH) using reverse transcription quantitative PCR (RT-qPCR).
• Analysis of gene expression stability using geNorm, BestKeeper, NormFinder, comparative ΔCt method, and RefFinder.

Main Results:

• All 13 tested genes exhibited relative expression stability in koala lymph node tissues.
• Tmem97 and Hmg20a were identified as the most stable reference genes across both koala populations.

Conclusions:

• Tmem97 and Hmg20a are recommended as reliable reference genes for gene expression studies in koala lymph nodes.
• These findings will improve the accuracy of immunological and disease-related research in koalas.