Reliable Reference Genes for the Quantification of MRNA in Human T-cells and PBMCs Stimulated with Live Influenza Virus

Overview

Journal BMC Immunol

Publisher Biomed Central

Specialty Allergy & Immunology

Date 2020 Feb 2

PMID 32005148

Citations 17

Authors

Justin G Roy

Janet E McElhaney

Chris P Verschoor

Affiliations

Soon will be listed here.

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.

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References

Chapman J, Waldenstrom J . With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies. PLoS One. 2015; 10(11):e0141853. PMC: 4640531. DOI: 10.1371/journal.pone.0141853. View

Steegenga W, Boekschoten M, Lute C, Hooiveld G, de Groot P, Morris T . Genome-wide age-related changes in DNA methylation and gene expression in human PBMCs. Age (Dordr). 2014; 36(3):9648. PMC: 4082572. DOI: 10.1007/s11357-014-9648-x. View

Harrison O, Moorjani N, Torrens C, Ohri S, Cagampang F . Endogenous Reference Genes for Gene Expression Studies on Bicuspid Aortic Valve Associated Aortopathy in Humans. PLoS One. 2016; 11(10):e0164329. PMC: 5058495. DOI: 10.1371/journal.pone.0164329. View

Thellin O, Elmoualij B, Heinen E, Zorzi W . A decade of improvements in quantification of gene expression and internal standard selection. Biotechnol Adv. 2009; 27(4):323-33. DOI: 10.1016/j.biotechadv.2009.01.010. View

Bustin S, Benes V, Nolan T, Pfaffl M . Quantitative real-time RT-PCR--a perspective. J Mol Endocrinol. 2005; 34(3):597-601. DOI: 10.1677/jme.1.01755. View

Ledderose C, Heyn J, Limbeck E, Kreth S . Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils. BMC Res Notes. 2011; 4:427. PMC: 3229292. DOI: 10.1186/1756-0500-4-427. View

Bas A, Forsberg G, Hammarstrom S, Hammarstrom M . Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes. Scand J Immunol. 2004; 59(6):566-73. DOI: 10.1111/j.0300-9475.2004.01440.x. View

Falsey A, Hennessey P, Formica M, Cox C, Walsh E . Respiratory syncytial virus infection in elderly and high-risk adults. N Engl J Med. 2005; 352(17):1749-59. DOI: 10.1056/NEJMoa043951. View

Jacob F, Guertler R, Naim S, Nixdorf S, Fedier A, Hacker N . Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines. PLoS One. 2013; 8(3):e59180. PMC: 3598660. DOI: 10.1371/journal.pone.0059180. View

10.

Zhou X, McElhaney J . Age-related changes in memory and effector T cells responding to influenza A/H3N2 and pandemic A/H1N1 strains in humans. Vaccine. 2011; 29(11):2169-77. PMC: 3057414. DOI: 10.1016/j.vaccine.2010.12.029. View

11.

Andersen C, Jensen J, Orntoft T . Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004; 64(15):5245-50. DOI: 10.1158/0008-5472.CAN-04-0496. View

12.

Bustin S, Benes V, Garson J, Hellemans J, Huggett J, Kubista M . The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009; 55(4):611-22. DOI: 10.1373/clinchem.2008.112797. View

13.

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3(7):RESEARCH0034. PMC: 126239. DOI: 10.1186/gb-2002-3-7-research0034. View

14.

Piehler A, Grimholt R, Ovstebo R, Berg J . Gene expression results in lipopolysaccharide-stimulated monocytes depend significantly on the choice of reference genes. BMC Immunol. 2010; 11:21. PMC: 2884165. DOI: 10.1186/1471-2172-11-21. View

15.

Haq K, Fulop T, Tedder G, Gentleman B, Garneau H, Meneilly G . Cytomegalovirus Seropositivity Predicts a Decline in the T Cell But Not the Antibody Response to Influenza in Vaccinated Older Adults Independent of Type 2 Diabetes Status. J Gerontol A Biol Sci Med Sci. 2016; 72(9):1163-1170. PMC: 5861868. DOI: 10.1093/gerona/glw216. View

16.

Dheda K, Huggett J, Chang J, Kim L, Bustin S, Johnson M . The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Anal Biochem. 2005; 344(1):141-3. DOI: 10.1016/j.ab.2005.05.022. View

17.

McElhaney J, Gentleman B . Cell-Mediated Immune Response to Influenza Using Ex Vivo Stimulation and Assays of Cytokine and Granzyme B Responses. Methods Mol Biol. 2015; 1343:121-41. DOI: 10.1007/978-1-4939-2963-4_11. View

18.

Oturai D, Sondergaard H, Bornsen L, Sellebjerg F, Romme Christensen J . Identification of Suitable Reference Genes for Peripheral Blood Mononuclear Cell Subset Studies in Multiple Sclerosis. Scand J Immunol. 2015; 83(1):72-80. DOI: 10.1111/sji.12391. View

19.

Chen I, Chou L, Chou S, Wang J, Stott J, Blanchard M . Selection of suitable reference genes for normalization of quantitative RT-PCR in peripheral blood samples of bottlenose dolphins (Tursiops truncatus). Sci Rep. 2015; 5:15425. PMC: 4614023. DOI: 10.1038/srep15425. View

20.

Kuchipudi S, Tellabati M, Nelli R, White G, Perez B, Sebastian S . 18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells. Virol J. 2012; 9:230. PMC: 3499178. DOI: 10.1186/1743-422X-9-230. View