Identification and validation of extracellular vesicle reference genes for the normalization of RT-qPCR data

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Extracellular vesicles (EVs) are crucial biomarkers and therapeutic agents, containing diverse nucleic acids.
• Accurate quantification of EV-associated RNA using reverse transcription-quantitative PCR (RT-qPCR) is hindered by the absence of reliable reference genes (RGs).

Purpose of the Study:

• To identify and validate universal, stably expressed reference genes for normalizing RT-qPCR analysis of EV-associated RNA.
• To establish SNRPG, OST4, TOMM7, and NOP10 as reliable RGs for EV research.

Main Methods:

• RT-qPCR was employed to assess the expression stability of candidate RGs in various cell lines, secreted EVs, and human biofluids.
• Enzymatic treatments and analysis of EV subpopulations were performed to confirm RG localization and stability.
• RefFinder analysis was used to compare the stability of novel RGs against established RGs.

Main Results:

• SNRPG, OST4, TOMM7, and NOP10 demonstrated stable expression across multiple cell lines, EVs (n=12), and human biofluids (n=3) under diverse conditions.
• These four RGs were confirmed to be present within EVs and stable across various EV subpopulations.
• SNRPG, OST4, TOMM7, and NOP10 exhibited superior stability compared to commonly used RGs like HMBS, YWHAZ, SDHA, and GAPDH.

Conclusions:

• SNRPG, OST4, TOMM7, and NOP10 are validated as universal and stable reference genes for EV-associated RNA analysis.
• These RGs facilitate accurate normalization in RT-qPCR, advancing EV research and clinical applications.
• The established RGs provide a foundation for standardized analysis of EV RNA cargo.