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      Identification of endogenous normalizing genes for expression studies in inguinal ring tissue for scrotal hernias in pigs

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          Abstract

          The use of reference genes is required for relative quantification in gene expression analysis and since the stability of these genes could be variable depending on the experimental design, it has become indispensable to test the reliability of endogenous genes. Therefore, this study evaluated 10 reference candidate genes in two different experimental conditions in order to obtain stable genes to be used as reference in expression studies related to scrotal hernias in pigs. Two independent experiments were performed: one with 30 days-old MS115 pigs and the other with 60 days-old Landrace pigs. The inguinal ring/canal was collected, frozen and further submitted to real-time PCR analysis (qPCR). For the reference genes stability evaluation, four tools were used: GeNorm in the SLqPCR, BestKeeper, NormFinder and Comparative CT. A general ranking was generated using the BruteAggreg function of R environment. In this study, the RPL19 was one of the most reliable endogenous genes for both experiments. The breed/age effects influenced the expression stability of candidate reference genes evaluated in the inguinal ring of pigs. Therefore, this study reinforces the importance of evaluating the stability of several endogenous genes previous their use, since a consensual set of reference genes is not easily obtained. Here, two sets of genes are recommended: RPL19, RPL32 and H3F3A for 30-days MS115 and PPIA and RPL19 for the 60 days-old Landrace pigs. This is the first study using the inguinal ring tissue and the results can be useful as an indicative for other studies working with gene expression in this tissue.

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

          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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            Reference genes in real-time PCR

            This paper aims to discuss various aspects of the use of reference genes in qPCR technique used in the thousands of present studies. Most frequently, these are housekeeping genes and they must meet several criteria so that they can lay claim to the name. Lots of papers report that in different conditions, for different organisms and even tissues the basic assumption—the constant level of the expression is not maintained for many genes that seem to be perfect candidates. Moreover, their transcription can not be affected by experimental factors. Sounds simple and clear but a great number of designed protocols and lack of consistency among them brings confusion on how to perform experiment properly. Since during selection of the most stable normalizing gene we can not use any reference gene, different ways and algorithms for their selection were developed. Such methods, including examples of best normalizing genes in some specific cases and possible mistakes are presented based on available sources. Numerous examples of reference genes applications, which are usually in too few numbers in relevant articles not allowing to make a solid fundament for a reader, will be shown along with instructive compilations to make an evidence for presented statements and an arrangement of future qPCR experiments. To include all the pitfalls and problems associated with the normalization methods there is no way not to begin from sample preparation and its storage going through candidate gene selection, primer design and statistical analysis. This is important because numerous short reviews available cover the topic only in lesser extent at the same time giving the reader false conviction of complete topic recognition.
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              Validation of housekeeping genes for normalizing RNA expression in real-time PCR.

              Analysis of RNA expression using techniques like real-time PCR has traditionally used reference or housekeeping genes to control for error between samples. This practice is being questioned as it becomes increasingly clear that some housekeeping genes may vary considerably in certain biological samples. We used real-time reverse transcription PCR (RT-PCR) to assess the levels of 13 housekeeping genes expressed in peripheral blood mononuclear cell culture and whole blood from healthy individuals and those with tuberculosis. Housekeeping genes were selected from conventionally used ones and from genes reported to be invariant in human T cell culture. None of the commonly used housekeeping genes [e.g., glyceraldehyde-phosphate-dehydrogenase (GAPDH)] were found to be suitable as internal references, as they were highly variable (>30-fold maximal variability). Furthermore, genes previously found to be invariant in human T cell culture also showed large variation in RNA expression (>34-fold maximal variability). Genes that were invariant in blood were highly variable in peripheral blood mononuclear cell culture. Our data show that RNA specifying human acidic ribosomal protein was the most suitable housekeeping gene for normalizing mRNA levels in human pulmonary tuberculosis. Validations of housekeeping genes are highly specific for a particular experimental model and are a crucial component in assessing any new model.
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                Author and article information

                Contributors
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Writing – original draft
                Role: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: Data curationRole: MethodologyRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                20 September 2018
                2018
                : 13
                : 9
                : e0204348
                Affiliations
                [1 ] Programa de Pós-graduação em Zootecnia, Centro de Educação Superior do Oeste (CEO), Universidade do Estado de Santa Catarina, UDESC, Chapecó, Santa Catarina, Brazil
                [2 ] Embrapa Suínos e Aves, Concórdia, Santa Catarina, Brazil
                [3 ] Universidade do Contestado, Concórdia, Santa Catarina, Brazil
                [4 ] Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil
                Universitat de Lleida, SPAIN
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-2645-4735
                Article
                PONE-D-18-11664
                10.1371/journal.pone.0204348
                6147718
                30235332
                95c20e4d-1a7c-4bcc-8627-502f0b24aad4
                © 2018 Lorenzetti et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 18 April 2018
                : 6 September 2018
                Page count
                Figures: 5, Tables: 3, Pages: 17
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100003593, Conselho Nacional de Desenvolvimento Científico e Tecnológico;
                Award ID: 476146/2013-5
                Award Recipient :
                This study was supported by project #476146/2013-5 from the National Council of Scientific and Technological Development (CNPq) to MCL in which she is a CNPq fellow. WR Lorenzetti was sponsored by a PROMOP/Udesc scholarship. IR Savoldi and KB do Carmo are recipients of a PIBIC/CNPq scholarship at Embrapa Swine and Poultry National Research Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Vertebrates
                Amniotes
                Mammals
                Swine
                Biology and Life Sciences
                Genetics
                Gene Expression
                Medicine and Health Sciences
                Diagnostic Medicine
                Signs and Symptoms
                Hernia
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Signs and Symptoms
                Hernia
                Biology and Life Sciences
                Agriculture
                Livestock
                Biology and life sciences
                Biochemistry
                Proteins
                DNA-binding proteins
                Biology and Life Sciences
                Genetics
                Gene Amplification
                Research and analysis methods
                Extraction techniques
                RNA extraction
                Physical Sciences
                Mathematics
                Applied Mathematics
                Algorithms
                Research and Analysis Methods
                Simulation and Modeling
                Algorithms
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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