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      Evaluation of reference genes for gene expression in red-tailed phascogale ( Phascogale calura) liver, lung, small intestine and spleen

      research-article
      , ,
      PeerJ
      PeerJ Inc.
      Immunity, Reference genes, Real-time PCR, Marsupial, qPCR, Housekeeping genes

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          Abstract

          Background

          Reference genes serve an important role as an endogenous control/standard for data normalisation in gene expression studies. Although reference genes have recently been suggested for marsupials, independent analysis of reference genes on different immune tissues is yet to be tested. Therefore, an assessment of reference genes is needed for the selection of stable, expressed genes across different marsupial tissues.

          Methods

          The study was conducted on red-tailed phascogales ( Phascogale calura) using five juvenile and five adult males. The stability of five reference genes (glyceraldehyde-3-phosphate dehydrogenase, GAPDH; β-actin, ACTB; 18S rRNA, 18S; 28S rRNA , 28S; and ribosomal protein L13A, RPL13A) was investigated using SYBR Green and analysed with the geNorm application available in qBase PLUS software.

          Results

          Gene stability for juvenile and adult tissue samples combined show that GAPDH was most stable in liver and lung tissue, and 18S in small intestine and spleen. While all reference genes were suitable for small intestine and spleen tissues, all reference genes except 28S were stable for lung and only 18S and 28S were stable for liver tissue. Separating the two age groups, we found that two different reference genes were considered stable in juveniles ( ACTB and GAPDH) and adults ( 18S and 28S), and RPL13A was not stable for juvenile small intestine tissue. Except for 28S, all reference genes were stable in juvenile and adult lungs, and all five reference genes were stable in spleen tissue.

          Discussion

          Based on expression stability, ACTB and GAPDH are suitable for all tissues when studying the expression of marsupials in two age groups, except for adult liver tissues. The expression stability between juvenile and adult liver tissue was most unstable, as the stable reference genes for juveniles and adults were different. Juvenile and adult lung, small intestine and spleen share similar stable reference genes, except for small intestine tissues where all reference genes were stable in adults but RPL13A was not suitable in juveniles.

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          Most cited references33

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          Housekeeping genes as internal standards: use and limits.

          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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            Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR

            Background Real-time quantitative PCR (qPCR) is a method for rapid and reliable quantification of mRNA transcription. Internal standards such as reference genes are used to normalise mRNA levels between different samples for an exact comparison of mRNA transcription level. Selection of high quality reference genes is of crucial importance for the interpretation of data generated by real-time qPCR. Results In this study nine commonly used reference genes were investigated in 17 different pig tissues using real-time qPCR with SYBR green. The genes included beta-actin (ACTB), beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L4 (RPL4), succinate dehydrogenase complex subunit A (SDHA), TATA box binding protein (TPB)and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ). The stability of these reference genes in different pig tissues was investigated using the geNorm application. The range of expression stability in the genes analysed was (from the most stable to the least stable): ACTB/RPL4, TBP, HPRT, HMBS, YWHAZ, SDHA, B2M and GAPDH. Conclusion Expression stability varies greatly between genes. ACTB, RPL4, TPB and HPRT1 were found to have the highest stability across tissues. Based on both expression stability and expression level, our data suggest that ACTB and RPL4 are good reference genes for high abundant transcripts while TPB and HPRT1 are good reference genes for low abundant transcripts in expression studies across different pig tissues.
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              beta-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels.

              The use of reverse transcription-polymerase chain reaction (RT-PCR) to measure mRNA levels has led to the common use of beta-actin and GAPDH housekeeping genes as denominators for comparison of samples. Expression of these genes is assumed to remain constant, so normalising for variations in processing and signal quantitation. However, it is well documented that beta-actin and GAPDH expression is upregulated with proliferation, activation, and differentiation. We hypothesised that airway samples which differ in their cellular profiles and activation status have different levels of expression of GAPDH and beta-actin. The mRNA for beta-actin, GAPDH, and interleukin (IL)-2 was measured in bronchoalveolar lavage (BAL) fluid cells and endobronchial biopsy tissue by competitive RT-PCR in a cross sectional study of 26 normal controls and 92 asthmatic subjects. For both BAL fluid cells and biopsy tissue, asthmatics overall had reduced expression of GAPDH and beta-actin mRNA. In asthmatic subjects not using inhaled corticosteroids (ICS), GAPDH mRNA levels in both BAL fluid and biopsy tissue, and beta-actin mRNA in BAL fluid cells were 10 times lower than samples from both normal controls and from asthmatic subjects using ICS. beta-Actin mRNA in biopsy specimens showed the same pattern of expression, but asthmatic subjects not using ICS were not significantly different from those receiving ICS treatment. IL-2 mRNA levels did not differ between the subject or treatment groups but, when expressed as a ratio with beta-actin, significant differences were seen. beta-Actin and GAPDH used as denominators of gene expression quantitation in asthma research can cause confounding. Housekeeping genes need careful validation before their use in such quantitative mRNA assays.
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                Author and article information

                Contributors
                Journal
                PeerJ
                PeerJ
                peerj
                peerj
                PeerJ
                PeerJ Inc. (San Francisco, USA )
                2167-8359
                13 October 2016
                2016
                : 4
                : e2552
                Affiliations
                [-1]School of Science and Health, Western Sydney University , Richmond, New South Wales, Australia
                Article
                2552
                10.7717/peerj.2552
                5068414
                e37d0593-e5fa-450d-9966-be35a1ab5df2
                ©2016 Ong et al.

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.

                History
                : 13 July 2016
                : 12 September 2016
                Funding
                Funded by: School of Science and Health, Western Sydney University
                This study and the Small Native Mammal Teaching and Research Facility were funded by the School of Science and Health, Western Sydney University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Genomics
                Molecular Biology

                immunity,reference genes,real-time pcr,marsupial,qpcr,housekeeping genes

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