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

TY - JOUR

T1 - Identification of Suitable Reference Genes for Peripheral Blood Mononuclear Cell Subset Studies in Multiple Sclerosis

AU - Oturai, Ditte Bang

AU - Søndergaard, H B

AU - Börnsen, L

AU - Sellebjerg, F

AU - Christensen, Jeppe Romme

N1 - © 2015 The Foundation for the Scandinavian Journal of Immunology.

PY - 2016/1/1

Y1 - 2016/1/1

N2 - Quantitative real-time PCR (qPCR) involves the need of a proper standard for normalizing the gene expression data. Different studies have shown the validity of reference genes to vary greatly depending on tissue, cell subsets and experimental context. This study aimed at the identification of suitable reference genes for qPCR studies using different peripheral blood cell subsets (whole blood (WB) cells, peripheral blood mononuclear cells (PBMCs) and PBMC subsets (CD4(+) T cells, CD8(+) T cells, NK cells, monocytes, B cells and dendritic cells) from healthy controls (HC), patients with relapsing-remitting multiple sclerosis (RRMS) and interferon-β-treated patients with RRMS (RRMS-IFN-β). Eight candidate reference genes (CASC3, EEF1A1, GAPDH, HPRT1, RPLP0, UBC, UBE2D2 and YWHAZ) were analysed using normfinder and genorm algorithms to identify the most stably expressed genes. We found reference gene expression varied most across cell subsets, and less variation between the donor groups. UBE2D2 was the most stably expressed gene across both HC and RRMS patients and across cell subsets, while UBC was the most stably expressed gene between HC, RRMS and RRMS-IFN-β patients. UBE2D2 and HPRT1 was the most stable combination for analyses of cell subsets between HC and RRMS patients, while the combination of UBC and YWHAZ was superior for analysis of cell subsets between HC, RRMS and RRMS-IFN-β groups. GAPDH was generally unsuitable for blood cell subset studies in multiple sclerosis. In conclusion, we found that blood cell subsets result in marked variation in reference gene expression, and we identified suitable reference genes for studies involving PBMC subsets, RRMS patients and interferon-β treatment.

AB - Quantitative real-time PCR (qPCR) involves the need of a proper standard for normalizing the gene expression data. Different studies have shown the validity of reference genes to vary greatly depending on tissue, cell subsets and experimental context. This study aimed at the identification of suitable reference genes for qPCR studies using different peripheral blood cell subsets (whole blood (WB) cells, peripheral blood mononuclear cells (PBMCs) and PBMC subsets (CD4(+) T cells, CD8(+) T cells, NK cells, monocytes, B cells and dendritic cells) from healthy controls (HC), patients with relapsing-remitting multiple sclerosis (RRMS) and interferon-β-treated patients with RRMS (RRMS-IFN-β). Eight candidate reference genes (CASC3, EEF1A1, GAPDH, HPRT1, RPLP0, UBC, UBE2D2 and YWHAZ) were analysed using normfinder and genorm algorithms to identify the most stably expressed genes. We found reference gene expression varied most across cell subsets, and less variation between the donor groups. UBE2D2 was the most stably expressed gene across both HC and RRMS patients and across cell subsets, while UBC was the most stably expressed gene between HC, RRMS and RRMS-IFN-β patients. UBE2D2 and HPRT1 was the most stable combination for analyses of cell subsets between HC and RRMS patients, while the combination of UBC and YWHAZ was superior for analysis of cell subsets between HC, RRMS and RRMS-IFN-β groups. GAPDH was generally unsuitable for blood cell subset studies in multiple sclerosis. In conclusion, we found that blood cell subsets result in marked variation in reference gene expression, and we identified suitable reference genes for studies involving PBMC subsets, RRMS patients and interferon-β treatment.

KW - Flow Cytometry

KW - Gene Expression Profiling

KW - Humans

KW - Immunologic Factors

KW - Interferon-beta

KW - Leukocytes, Mononuclear

KW - Multiple Sclerosis, Relapsing-Remitting

KW - Real-Time Polymerase Chain Reaction

KW - Reference Standards

KW - Transcriptome

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1111/sji.12391

DO - 10.1111/sji.12391

M3 - Journal article

C2 - 26395032

SN - 0301-6323

VL - 83

SP - 72

EP - 80

JO - Scandinavian Journal of Immunology, Supplement

JF - Scandinavian Journal of Immunology, Supplement

IS - 1

ER -