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

Abstract

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-b-treated patients with RRMS (RRMS-IFN-b). 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-b 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-b 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-b treatment.

Validation of qPCR reference genes in lymphocytes from patients with amyotrophic lateral sclerosis

“…Although there is a number of papers concerning the validation of ICG in lymphocytes [42, 43], there are no reports addressing this issue in LCLs. Moreover, to our knowledge, there have been no studies comparing the stability of ICG between the fresh PBMCs and LCLs obtained from the same individuals.…”

Free Circulating miRNAs Measurement in Clinical Settings

“…Conversely to what many authors advise, Falkenberg and coauthors demonstrated that the use of a single reference gene is sufficient when the most stable is selected, rather than using multiple reference genes [66]. Similarly, Oturai and colleagues [70] identified the suitable reference genes using different peripheral blood cell populations, such as whole blood cells, PBMC, and PBMC-derived subsets of cells (CD4 þ T cells, CD8 þ T cells, NK cells, monocytes, B cells, and dendritic cells) obtained from healthy subjects, patients affected by multiple sclerosis (MS), and interferon b (IFNb)-treated MS patients. Out of eight candidate reference genes, the authors identified ubiquitin-conjugating enzyme E2D2 (UBE2D2) as the best candidate for normalizing samples from control and MS subjects and all cell subsets, while to compare healthy subjects, MS, and IFNb-treated MS patients, ubiquitin C (UBC) gene was the best normalizer across all the cell subsets.…”

“…Out of eight candidate reference genes, the authors identified ubiquitin-conjugating enzyme E2D2 (UBE2D2) as the best candidate for normalizing samples from control and MS subjects and all cell subsets, while to compare healthy subjects, MS, and IFNb-treated MS patients, ubiquitin C (UBC) gene was the best normalizer across all the cell subsets. Additionally, they also identified the best combination of two reference genes to normalize either control and MS groups, which were UBE2D2 and HPRT, or controls, MS, and IFNb-treated MS groups, which were UBC and Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta-polypeptide (YWHAZ) [70]. Noteworthy, reference genes that show a high variability of expression when considered as single normalizer might be optimal in combination with other reference gene(s).…”

Tryptophan and arginine catabolic enzymes and regulatory cytokines in clinically isolated syndrome and multiple sclerosis

“…For analysis of cultured cells, probes were for TNF, IL-1B, IL-6, IL-10, TGFB, cyclooxygenase 2 (COX2), IDO1, ARG1 and UBE2D2. UBE2D2 was used as the housekeeping gene because in a comparison with seven other potential housekeeping genes, it was the most stably expressed gene in cell subsets from HCs and those with relapsing-remitting MS. 31 All samples were loaded onto a 96-well plate that contained a nonreverse transcriptase control and a no template control. An aliquot from a large cDNA preparation (frozen as multiple vials) was added to every plate for UBE2D2 measurement and used as a PCR quality control.…”