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Reliable reference genes for the quantification of mRNA in human T-cells and PBMCs stimulated with live influenza virus
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Reliable reference genes for the quantification of mRNA in human T-cells and PBMCs stimulated with live influenza virus
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Journal Article
Reliable reference genes for the quantification of mRNA in human T-cells and PBMCs stimulated with live influenza virus
2020
Overview
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.
Publisher
BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
Subject
/ Age
/ Biomedical and Life Sciences
/ Cytokines and Growth Factors
/ Enzymes
/ Genes
/ Glyceraldehyde-3-phosphate dehydrogenase
/ Human peripheral blood mononuclear cells
/ Humans
/ Leukocytes, Mononuclear - immunology
/ Methods
/ Nature
/ Orthomyxoviridae - immunology
/ Peripheral blood mononuclear cells
/ Real-Time Polymerase Chain Reaction
/ Ribosomal Proteins - genetics
/ RNA
/ Software
/ T cells
/ Ubiquitin-conjugating enzyme
/ Ubiquitin-Conjugating Enzymes - genetics
/ Vaccine
