Internal reference genes with the potential for normalizing quantitative PCR results for oral fluid specimens

Area of Science:

• Biotechnology
• Molecular Biology
• Diagnostic Medicine

Background:

• Real-time quantitative polymerase chain reaction (qPCR) is utilized in basic research and diagnostic medicine.
• qPCR in oral fluid specimens is employed to assess gene expression and detect infections.
• Normalization using reference genes is crucial for qPCR accuracy, controlling for sample variations.

Purpose of the Study:

• To review the use of internal reference genes in qPCR assays for oral fluid specimens.
• To identify the extent to which normalization strategies are employed in recent human and veterinary studies.
• To highlight the implications of lacking validated reference genes for oral fluid qPCR.

Main Methods:

• Systematic review of recent publications in human and veterinary medicine utilizing qPCR on oral fluid specimens.
• Analysis of reported methodologies, specifically focusing on the use of internal reference genes for normalization.
• Categorization of studies based on the application of endogenous or exogenous reference genes.

Main Results:

• A significant majority of reviewed studies did not report the use of internal reference genes for oral fluid qPCR.
• 52.9% of animal studies and 57.0% of human studies omitted internal reference gene normalization.
• The application and validation of endogenous reference genes in oral fluid specimens remain underexplored.

Conclusions:

• The absence of validated internal reference genes in oral fluid specimens compromises the reliability and reproducibility of qPCR assays.
• Addressing the lack of suitable reference genes is essential for advancing the utility of oral fluid qPCR in research and diagnostics.
• Further research is needed to identify and validate appropriate reference genes for oral fluid matrix normalization.