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.

Keywords: Housekeeping genes; Human peripheral blood mononuclear cells; Influenza; Quantitative PCR; Reference genes; T-cells.

Fig. 1

Cq values for candidate reference genes in stimulated and unstimulated PBMCs and T-cells. Mean quantification cycle (Cq) values are presented for both unstimulated and influenza A/Victoria/375 stimulated donor (n = 10) PBMCs (a) and CD3⁺ T-cells (b). Unstimulated controls are presented by the black, filled circles, and the paired stimulated samples are presented by clear circles. Statistical analysis was performed by paired t-test or Wilcoxon signed rank test. P-values are presented between indicated comparisons. Data from 10 young and old donors is presented