Identification of Suitable Reference Genes for Peripheral Blood Mononuclear Cell Subset Studies in Multiple Sclerosis
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
Standard
Identification of Suitable Reference Genes for Peripheral Blood Mononuclear Cell Subset Studies in Multiple Sclerosis. / Oturai, Ditte Bang; Søndergaard, H B; Börnsen, L; Sellebjerg, F; Christensen, Jeppe Romme.
I: Scandinavian Journal of Immunology, Bind 83, Nr. 1, 01.2016, s. 72-80.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
Harvard
APA
Vancouver
Author
Bibtex
}
RIS
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
Y1 - 2016/1
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
VL - 83
SP - 72
EP - 80
JO - Scandinavian Journal of Immunology, Supplement
JF - Scandinavian Journal of Immunology, Supplement
SN - 0301-6323
IS - 1
ER -
ID: 179159992