Reliable reference genes for the quantification of mRNA in human T-cells and PBMCs stimulated with live influenza virus

Abstract

Background: Quantitative PCR (qPCR) is a powerful tool that is particularly well-suited to measure mRNA levels in clinical samples, especially those with relatively low cell counts. However, a caveat of this approach is that reliable, stably expressed reference (housekeeping) genes are vital in order to ensure reproducibility and appropriate biological inference. In this study, we evaluated the expression stability of six reference genes in peripheral blood mononuclear cells (PBMCs) and isolated CD3 + T-cells from young and old adults (n = 10), following ex vivo stimulation with mock (unstimulated) or live influenza virus. Our genes included: β-actin (ACTB), glyercaldehyde-3-phostphate dehydrogenase (GAPDH), ribosomal protein L13a (RPL13a), ribosomal protein S18 (RPS18), succinate dehydrogenase complex flavoprotein subunit A (SDHA), and ubiquitin-conjugating enzyme E2D2 (UBE2D2). Results: Reference gene expression varied significantly depending on cell type and stimulation conditions, but not age. Using the comparative ΔCt method, and the previously published software BestKeeper, NormFinder, and geNorm, we show that in PBMCs and T-cells, UBE2D2 and RPS18 were the most stable reference genes, followed by ACTB; however, the expression of UBE2D2 and RPS18 was found to increase with viral stimulation in isolated T-cells, while ACTB expression did not change significantly. No age-related differences in stability were observed for any gene Conclusions: This study suggests the use of a combination of UBE2D2, RPS18, and ACTB for the study of influenza responses in PBMCs and T-cells, although ACTB alone may be the most optimal choice if choosing to compare target gene expression before and after viral stimulation. Both GAPDH and RPL13a were found to be poor reference genes and should be avoided for studies of this nature.

ARPC5 Isoforms and Their Regulation by Calcium-Calmodulin-N-WASP Drive Distinct Arp2/3-dependent Actin Remodeling Events in CD4 T Cells

“…This induction of protein levels by T cell activation was paralleled by induction of mRNA expression (Fig 2B : absolute values; Fig. 2C: relative to GAPDH; note that housekeeping genes are also subject to regulation by T cell activation (Sousa, Simi et al 2019, Roy, McElhaney et al 2020, Subbannayya, Haug et al 2020). CD4 T cells thus express all ARP2/3 complex subunits isoforms and T cell activation increases Arp2/3 protein levels.…”

Dysregulation of phospholipase and cyclooxygenase expression is involved in Schizophrenia

“…The primer sequences used to amplify the target genes are listed in Supplementary Table 1. Two housekeeping genes— ACTB and SDHA —were used in the experiments [21] , and the stability was evaluated. SDHA were chosen as the internal controls used for quantitative analysis.…”

Identification of susceptibility loci for adverse events following COVID-19 vaccination in the Japanese population: A web-based genome-wide association study

“…Regarding other genes, increased NOTCH4 expression in circulating regulatory T cells in COVID-19 patients was associated with disease severity and predicted mortality 25 . The expression of RPS18 was previously found to be increased in isolated T-cells on stimulation with the live influenza virus 26 . A SNP in the BAK1 and A haplotype of MICB was associated with dengue hemorrhagic fever caused by dengue virus 27,28 .…”