Identification of stable reference genes in plasma astrocyte-derived exosomal mRNA for RT-qPCR analysis in Alzheimer's disease.

Accurate quantification of relative gene expression by RT-qPCR requires the selection of appropriate reference genes for data normalization. Recently, extracellular vesicle components, particularly exosomal RNAs, have recently emerged as promising biomarkers for Alzheimer's disease (AD). Among these, mRNAs from astrocyte-derived exosomes (ADEs) hold particular significance for AD diagnosis and prognosis due to their high in vivo stability and capacity to cross the blood-brain barrier, thereby faithfully reflecting cerebral pathological changes in AD patients. However, suitable reference genes for normalizing target gene expression in ADEs have not been systematically identified. In this study, we comprehensively evaluated the expression stability of candidate reference genes in plasma ADEs across cognitively unimpaired (CU), mild cognitive impairment (MCI), and AD patients using five robust algorithms, including Genorm, Bestkeeper, ΔCt method, Normfinder, and RefFinder. Our results revealed that YWHAE and RNPS1 exhibited superior expression stability across all three groups, whereas GAPDH, a conventionally used reference gene, demonstrated inadequate stability. Collectively, our work provides the first systematic validation of reference genes for mRNA quantification in plasma ADEs, establishing a methodological foundation for ADE-based AD biomarker development.