Identification of Suitable Reference Genes for Peripheral Blood Mononuclear Cell Subset Studies in Multiple Sclerosis

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• Quantitative real-time PCR (qPCR) requires stable reference genes for accurate gene expression normalization.
• Reference gene stability is known to vary significantly with tissue type, cell subsets, and experimental conditions.

Purpose of the Study:

• To identify optimal reference genes for qPCR in various peripheral blood cell subsets.
• To evaluate reference gene stability in healthy controls, relapsing-remitting multiple sclerosis (RRMS) patients, and RRMS patients treated with interferon-beta (IFN-β).

Main Methods:

• Analysis of eight candidate reference genes (CASC3, EEF1A1, GAPDH, HPRT1, RPLP0, UBC, UBE2D2, YWHAZ) using NormFinder and Genorm algorithms.
• Assessment across whole blood (WB), peripheral blood mononuclear cells (PBMCs), and specific PBMC subsets (CD4+ T cells, CD8+ T cells, NK cells, monocytes, B cells, dendritic cells).
• Comparison between healthy controls (HC), RRMS patients, and RRMS-IFN-β patients.

Main Results:

• Reference gene expression stability varied most across cell subsets, with less variation between patient groups.
• UBE2D2 demonstrated the highest stability across HC and RRMS patients and cell subsets.
• UBC was the most stable gene when comparing HC, RRMS, and RRMS-IFN-β patients.
• Specific gene combinations (UBE2D2/HPRT1 and UBC/YWHAZ) were identified as superior for different comparison groups.
• GAPDH was found to be unsuitable for multiple sclerosis blood cell subset studies.

Conclusions:

• Peripheral blood cell subsets significantly influence reference gene expression stability in qPCR.
• UBE2D2 and UBC are recommended as highly stable reference genes for specific experimental contexts in multiple sclerosis research.
• Careful selection of reference genes is essential for reliable gene expression analysis in immunological studies.