Validation of the Reference Genes for the Gene Expression Studies in Chicken DT40 Cell Line
The selection of a suitable reference gene assures a reliable gene expression analysis when using the qPCR method. Normalization of the reaction is based on the basic metabolism genes. These genes show a constant, unregulated expression in all cells and function throughout their lifetime. In the cur...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-03-01
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| Series: | Genes |
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| Online Access: | https://www.mdpi.com/2073-4425/11/4/372 |
| _version_ | 1797571934103273472 |
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| author | Aleksandra Dunislawska Anna Slawinska Maria Siwek |
| author_facet | Aleksandra Dunislawska Anna Slawinska Maria Siwek |
| author_sort | Aleksandra Dunislawska |
| collection | DOAJ |
| description | The selection of a suitable reference gene assures a reliable gene expression analysis when using the qPCR method. Normalization of the reaction is based on the basic metabolism genes. These genes show a constant, unregulated expression in all cells and function throughout their lifetime. In the current study, seven reference gene candidates were screened using RT-qPCR, to determine the best-matched pair of reference genes in the chicken DT40 cell line. The DT40 was derived from bursal lymphoma cells that were subjected to RAV-1 bird retroviral infection. It is a simplified in vitro model that allows tracking the direct interaction of stimulants on the lymphoid population and profiling of the hepatocellular B cell transcriptome. The reference gene analysis was carried out using statistical tools integrating four independent methods—geNorm, Best Keeper, NormFinder, delta Ct and RefFinder. Based on the selected reference genes, the relative gene expression analysis was done using the ddCt method. Complete relative gene expression study on a panel of the target genes revealed that proper selection of reference genes depending on the tissue eliminate decreases in data quality. The <i>SDHA</i> and <i>RPL4</i> genes constitute stable internal controls as reference genes when analyzing gene expression in the DT40 cell line. |
| first_indexed | 2024-03-10T20:47:39Z |
| format | Article |
| id | doaj.art-2d55becd1b1a440cbbb9027e7ae2bc20 |
| institution | Directory Open Access Journal |
| issn | 2073-4425 |
| language | English |
| last_indexed | 2024-03-10T20:47:39Z |
| publishDate | 2020-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Genes |
| spelling | doaj.art-2d55becd1b1a440cbbb9027e7ae2bc202023-11-19T20:08:59ZengMDPI AGGenes2073-44252020-03-0111437210.3390/genes11040372Validation of the Reference Genes for the Gene Expression Studies in Chicken DT40 Cell LineAleksandra Dunislawska0Anna Slawinska1Maria Siwek2Department of Animal Biotechnology and Genetics, University of Science and Technology, 85-084 Bydgoszcz, PolandDepartment of Animal Biotechnology and Genetics, University of Science and Technology, 85-084 Bydgoszcz, PolandDepartment of Animal Biotechnology and Genetics, University of Science and Technology, 85-084 Bydgoszcz, PolandThe selection of a suitable reference gene assures a reliable gene expression analysis when using the qPCR method. Normalization of the reaction is based on the basic metabolism genes. These genes show a constant, unregulated expression in all cells and function throughout their lifetime. In the current study, seven reference gene candidates were screened using RT-qPCR, to determine the best-matched pair of reference genes in the chicken DT40 cell line. The DT40 was derived from bursal lymphoma cells that were subjected to RAV-1 bird retroviral infection. It is a simplified in vitro model that allows tracking the direct interaction of stimulants on the lymphoid population and profiling of the hepatocellular B cell transcriptome. The reference gene analysis was carried out using statistical tools integrating four independent methods—geNorm, Best Keeper, NormFinder, delta Ct and RefFinder. Based on the selected reference genes, the relative gene expression analysis was done using the ddCt method. Complete relative gene expression study on a panel of the target genes revealed that proper selection of reference genes depending on the tissue eliminate decreases in data quality. The <i>SDHA</i> and <i>RPL4</i> genes constitute stable internal controls as reference genes when analyzing gene expression in the DT40 cell line.https://www.mdpi.com/2073-4425/11/4/372housekeeping geneschicken B cellsin vitroqPCRquantitative gene expression |
| spellingShingle | Aleksandra Dunislawska Anna Slawinska Maria Siwek Validation of the Reference Genes for the Gene Expression Studies in Chicken DT40 Cell Line Genes housekeeping genes chicken B cells in vitro qPCR quantitative gene expression |
| title | Validation of the Reference Genes for the Gene Expression Studies in Chicken DT40 Cell Line |
| title_full | Validation of the Reference Genes for the Gene Expression Studies in Chicken DT40 Cell Line |
| title_fullStr | Validation of the Reference Genes for the Gene Expression Studies in Chicken DT40 Cell Line |
| title_full_unstemmed | Validation of the Reference Genes for the Gene Expression Studies in Chicken DT40 Cell Line |
| title_short | Validation of the Reference Genes for the Gene Expression Studies in Chicken DT40 Cell Line |
| title_sort | validation of the reference genes for the gene expression studies in chicken dt40 cell line |
| topic | housekeeping genes chicken B cells in vitro qPCR quantitative gene expression |
| url | https://www.mdpi.com/2073-4425/11/4/372 |
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