scholarly journals Validation of the Reference Genes for the Gene Expression Studies in Chicken DT40 Cell Line

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 372
Author(s):  
Aleksandra Dunislawska ◽  
Anna Slawinska ◽  
Maria Siwek
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Relative Gene Expression ◽  
Dt40 Cell ◽  
Relative Gene ◽  
Chicken Dt40 Cell ◽  
Selection Of

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 SDHA and RPL4 genes constitute stable internal controls as reference genes when analyzing gene expression in the DT40 cell line.

Selection of reference genes for the study of relative gene expression during development of Litopenaeus vannamei larvae

2020 ◽  
Vol 51 (7) ◽  
pp. 2997-3006
Author(s):  
Laura Alvarez‐Lee ◽  
Alejandra García‐Gasca ◽  
Sergio Martínez‐Díaz ◽  
Neftalí Gutiérrez‐Rivera
Keyword(s):  
Gene Expression ◽  
Litopenaeus Vannamei ◽  
Reference Genes ◽  
Relative Gene Expression ◽  
Relative Gene ◽  
Selection Of

scholarly journals Selecting Suitable Reference Genes for qPCR Normalization: A Comprehensive Analysis in MCF-7 Breast Cancer Cell Line

2020 ◽  
Author(s):  
Nityanand Jain ◽  
Dina Nitisa ◽  
Valdis Pirsko ◽  
Inese Cakstina
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Reference Gene ◽  
Reference Genes ◽  
Cancer Cell Line ◽  
Internal Controls ◽  
Reference Gene Expression ◽  
Qpcr Normalization ◽  
Suitable Reference ◽  
Mcf 7

Abstract BackgroundMCF-7 breast cancer cell line is undoubtedly amongst the most extensively studied patient-derived research models, providing pivotal results that have over the decades translated to constantly improving patient care. Many research groups, have previously identified suitable reference genes for qPCR normalization in MCF-7 cell line. However, over the course of identification of suitable reference genes, a comparative analysis comprising these genes together in a single study have not been reported. Furthermore, the expression dynamics of these reference genes within sub-clones cultured over multiple passages (p) has attracted limited attention from research groups. Therefore, we investigated the expression dynamics of 12 previously suggested reference genes within two sub-clones (culture A1 and A2) cultured identically over multiple passages. Additionally, the effect of nutrient stress on reference gene expression was examined to devise an evidence-based recommendation of the least variable reference genes that could be employed in future gene expression studies.ResultsThe analysis revealed the presence of differential reference gene expression within the sub-clones of MCF-7. In culture A1, GAPDH-CCSER2 were identified as the least variable reference gene pair while for culture A2, GAPDH-RNA28S was identified. However, upon validation using genes of interest, both these pairs were found to be unsuitable control pairs. Normalization of AURKA and KRT19 with triplet pair GAPDH-PCBP1-CCSER2 yielded successful results. The triplet also proved its capability to handle variations arising from nutrient stress.ConclusionsThe variance in expression behavior amongst sub-clones highlights the potential need for exercising caution while selecting reference genes for MCF-7. GAPDH-PCBP1-CCSER2 triplet offers a reliable alternative to otherwise traditionally used internal controls for optimizing intra- and inter-assay gene expression differences. Furthermore, we suggest avoiding the use of ACTB, GAPDH and PGK1 as single internal controls.

scholarly journals Expression Stability of Internal Reference Gene in Response to Trichoderma polysporum Infection in Avena fatua L.

2021 ◽  
Author(s):  
Haixia Zhu ◽  
Yongqiang Ma ◽  
Liang Cheng
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Expression Analysis ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Avena Fatua ◽  
Expression Stability ◽  
Internal Reference ◽  
Qpcr Analysis ◽  
Suitable Combination

Abstract In order to construct a RT-qPCR system suitable for response of Avena fatua L. to Trichoderma polysporum , and screen stable internal reference genes, GeNorm, NormFinder, BestKeeper and RefFinde were used to perform SYBR Green-based RT-qPCR analysis on 8 candidate internal reference genes ( 18S , 28S , TUA , UBC , ACT , GAPDH , TBP and EF-1 ) in A. fatua samples after inoculation of T. polysporum Strain HZ-31. The results showed that TBP , 18S and UBC were the most stable internal reference genes, TBP and TUA , TBP and GAPDH , 18S and TBP , UBC and 18S were the most suitable combination of the two internal reference genes, which could be used as the internal reference genes for functional gene expression analysis during the interaction between T. polysporum and A. fatua .

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