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      Selection of a suitable reference gene for quantitative gene expression in mouse lymph nodes after vaccination

      research-article
      Author(s): ,
      Publication date (Electronic):
      Journal: BMC Research Notes
      Publisher: BioMed Central
      Keywords: DTaP, Lymph nodes, Mouse, qPCR, Reference gene, Ubc

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          Abstract

          Background

          The quantification of gene expression is an important tool in the evaluation of the immune response to vaccines. Reliable reference genes for gene expression studies in mouse draining lymph nodes after vaccination have not been reported.

          Results

          The utility of seven potential reference genes was investigated using commercially available Taq-man primer/probe mixes. Results were evaluated with RefFinder, a web-based program including multiple algorithm methods such as geNorm, NormFinder, BestKeeper and the comparative delta-Ct. Further assessment was done by applying the candidate reference genes in relative expression calculations with genes related to the magnitude and longevity of the humoral immune responses. The ubiquitin C gene, Ubc, was found to be the most reliable reference gene when validated with well-known genes that are expressed at relatively low levels after vaccination. The optimal time of sample collection varied depending on the function of the target genes.

          Conclusions

          This study identified Ubc as the most reliable reference gene and provides useful information for studies examining immunological gene expression in the draining lymph nodes after vaccination in mice.

          Electronic supplementary material

          The online version of this article (10.1186/s13104-017-3005-y) contains supplementary material, which is available to authorized users.

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          Most cited references23

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          Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR

          Nicholas Silver, Steve Best, Jie Jiang … (2006)
          Background Control genes, which are often referred to as housekeeping genes, are frequently used to normalise mRNA levels between different samples. However, the expression level of these genes may vary among tissues or cells and may change under certain circumstances. Thus, the selection of housekeeping genes is critical for gene expression studies. To address this issue, 7 candidate housekeeping genes including several commonly used ones were investigated in isolated human reticulocytes. For this, a simple ΔCt approach was employed by comparing relative expression of 'pairs of genes' within each sample. On this basis, stability of the candidate housekeeping genes was ranked according to repeatability of the gene expression differences among 31 samples. Results Initial screening of the expression pattern demonstrated that 1 of the 7 genes was expressed at very low levels in reticulocytes and was excluded from further analysis. The range of expression stability of the other 6 genes was (from most stable to least stable): GAPDH (glyceraldehyde 3-phosphate dehydrogenase), SDHA (succinate dehydrogenase), HPRT1 (hypoxanthine phosphoribosyl transferase 1), HBS1L (HBS1-like protein) and AHSP (alpha haemoglobin stabilising protein), followed by B2M (beta-2-microglobulin). Conclusion Using this simple approach, GAPDH was found to be the most suitable housekeeping gene for expression studies in reticulocytes while the commonly used B2M should be avoided.
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            Systems biology approach predicts immunogenicity of the yellow fever vaccine in humans.

            Troy Querec, Rama Akondy, Eva K. Lee … (2009)
            A major challenge in vaccinology is to prospectively determine vaccine efficacy. Here we have used a systems biology approach to identify early gene 'signatures' that predicted immune responses in humans vaccinated with yellow fever vaccine YF-17D. Vaccination induced genes that regulate virus innate sensing and type I interferon production. Computational analyses identified a gene signature, including complement protein C1qB and eukaryotic translation initiation factor 2 alpha kinase 4-an orchestrator of the integrated stress response-that correlated with and predicted YF-17D CD8(+) T cell responses with up to 90% accuracy in an independent, blinded trial. A distinct signature, including B cell growth factor TNFRS17, predicted the neutralizing antibody response with up to 100% accuracy. These data highlight the utility of systems biology approaches in predicting vaccine efficacy.
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              Housekeeping genes as internal standards: use and limits.

              O Thellin, W Zorzi, B Lakaye … (1999)
              Quantitative studies are commonly realised in the biomedical research to compare RNA expression in different experimental or clinical conditions. These quantifications are performed through their comparison to the expression of the housekeeping gene transcripts like glyceraldehyde-3-phosphate dehydrogenase (G3PDH), albumin, actins, tubulins, cyclophilin, hypoxantine phsophoribosyltransferase (HRPT), L32. 28S, and 18S rRNAs are also used as internal standards. In this paper, it is recalled that the commonly used internal standards can quantitatively vary in response to various factors. Possible variations are illustrated using three experimental examples. Preferred types of internal standards are then proposed for each of these samples and thereafter the general procedure concerning the choice of an internal standard and the way to manage its uses are discussed.
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                Author and article information

                Contributors
                Yung-Yi C. Mosley: chen37@purdue.edu
                Harm HogenEsch: hogenesch@purdue.edu
                Journal
                Journal ID (nlm-ta): BMC Res Notes
                Journal ID (iso-abbrev): BMC Res Notes
                Title: BMC Research Notes
                Publisher: BioMed Central (London )
                ISSN (Electronic): 1756-0500
                Publication date (Electronic): 6 December 2017
                Publication date PMC-release: 6 December 2017
                Publication date Collection: 2017
                Volume: 10
                Electronic Location Identifier: 689
                Affiliations
                ISNI 0000 0004 1937 2197, GRID grid.169077.e, Department of Comparative Pathobiology, College of Veterinary Medicine, , Purdue University, ; 725 Harrison Street, West Lafayette, IN 47907 USA
                Article
                Publisher ID: 3005
                DOI: 10.1186/s13104-017-3005-y
                PMC ID: 5718095
                PubMed ID: 29208024
                SO-VID: 496d378d-84de-433d-a794-ad042c1eabce
                Copyright © © The Author(s) 2017
                License:

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                Date received : 29 September 2016
                Date accepted : 28 November 2017
                Funding
                Funded by: the Hayward Genetics Foundation and the BMWLR Foundation
                Categories
                Subject: Research Article
                Custom metadata
                issue-copyright-statement © The Author(s) 2017

                ScienceOpen disciplines: Medicine
                Keywords: dtap,lymph nodes,mouse,qpcr,reference gene,ubc
                Data availability:
                ScienceOpen disciplines: Medicine
                Keywords: dtap, lymph nodes, mouse, qpcr, reference gene, ubc

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