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 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.

Stable reference genes in granulosa cells of bovine dominant follicles during follicular growth, FSH stimulation and maternal aging

2016 ◽  
Vol 28 (6) ◽  
pp. 795 ◽  
Author(s):  
Muhammad Irfan-ur-Rehman Khan ◽  
Fernanda Caminha Faustino Dias ◽  
Isabelle Dufort ◽  
Vikram Misra ◽  
Marc-Andre Sirard ◽  
...  
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Reference Genes ◽  
Geometric Mean ◽  
Mrna Levels ◽  
Relative Gene Expression ◽  
Single Reference Gene ◽  
Aging Study ◽  
Maternal Aging ◽  
Relative Gene

The aim of the present study was to determine a set of reference genes in granulosa cells of dominant follicles that are suitable for relative gene expression analyses during maternal and follicular aging. Granulosa cells of growing and preovulatory dominant follicles were collected from aged and young cows (maternal aging study) and from FSH-stimulated follicles developing under different durations of FSH treatment (follicular aging study). The mRNA levels of the two commonly used reference genes (GAPDH, ACTB) and four novel genes (UBE2D2, EIF2B2, SF3A1, RNF20) were analysed using cycle threshold values. Results revealed that mRNA levels of GAPDH, ACTB, EIF2B2, RNF20, SF3A1 and UBE2D2 were similar (P > 0.05) between dominant follicle type, age and among follicles obtained after FSH-stimulation, but differed (P = 0.005) due to mRNA processing (i.e. with versus without amplification). The stability of reference genes was analysed using GeNorm, DeltaCT and NormFinder programs and comprehensive ranking order was determined using RefFinder. The mRNA levels of GAPDH and ACTB were less stable than those of UBE2D2 and EIF2B2. The geometric mean of multiple genes (UBE2D2, EIF2B2, GAPDH and SF3A1) is a more appropriate reference control than the use of a single reference gene to compare relative gene expression among dominant and FSH-stimulated follicles during maternal and/or follicular aging studies.

scholarly journals Validation of endogenous reference genes in rat cerebral cortex for RT-qPCR analyses in developmental toxicity studies

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7181 ◽  
Author(s):  
Louise Ramhøj ◽  
Marta Axelstad ◽  
Terje Svingen
Keyword(s):  
Gene Expression ◽  
Cerebral Cortex ◽  
Reference Genes ◽  
In Utero ◽  
Rat Cerebral Cortex ◽  
In Utero Exposure ◽  
Relative Gene Expression ◽  
Endogenous Reference ◽  
Relative Gene ◽  
Endogenous Reference Genes

Relative gene expression data obtained from quantitative RT-PCR (RT-qPCR) experiments are dependent on appropriate normalization to represent true values. It is common to use constitutively expressed endogenous reference genes (RGs) for normalization, but for this strategy to be valid the RGs must be stably expressed across all the tested samples. Here, we have tested 10 common RGs for their expression stability in cerebral cortex from young rats after in utero exposure to thyroid hormone (TH) disrupting compounds. We found that all 10 RGs were stable according to the three algorithms geNorm, NormFinder and BestKeeper. The downstream target gene Pvalb was significantly downregulated in brains from young rats after in utero exposure to propylthiouracil (PTU), a medicinal drug inhibiting TH synthesis. Similar results were obtained regardless of which of the 10 RGs was used for normalization. Another potential gene affected by developmental TH disruption, Dio2, was either not affected, or significantly upregulated about 1.4-fold, depending on which RG was used for normalization. This highlights the importance of carefully selecting correct RGs for normalization and to take into account the sensitivity of the RT-qPCR method when reporting on changes to gene expression that are less than 1.5-fold. For future studies examining relative gene expression in rat cerebral cortex under toxicological conditions, we recommend using a combination of either Rps18/Rpl13a or Rps18/Ubc for normalization, but also continuously monitor any potential regulation of the RGs themselves following alterations to study protocols.

scholarly journals Spatio-temporal selection of reference genes in the two congeneric species of Glycyrrhiza

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuping Li ◽  
Xiaoju Liang ◽  
Xuguo Zhou ◽  
Yu An ◽  
Ming Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Developmental Stage ◽  
Reference Genes ◽  
Developmental Stages ◽  
Individual Factors ◽  
Congeneric Species ◽  
Spatio Temporal ◽  
The Stability ◽  
Different Tissues ◽  
Selection Of

AbstractGlycyrrhiza, a genus of perennial medicinal herbs, has been traditionally used to treat human diseases, including respiratory disorders. Functional analysis of genes involved in the synthesis, accumulation, and degradation of bioactive compounds in these medicinal plants requires accurate measurement of their expression profiles. Reverse transcription quantitative real-time PCR (RT-qPCR) is a primary tool, which requires stably expressed reference genes to serve as the internal references to normalize the target gene expression. In this study, the stability of 14 candidate reference genes from the two congeneric species G. uralensis and G. inflata, including ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2, were evaluated across different tissues and throughout various developmental stages. More importantly, we investigated the impact of interactions between tissue and developmental stage on the performance of candidate reference genes. Four algorithms, including geNorm, NormFinder, BestKeeper, and Delta Ct, were used to analyze the expression stability and RefFinder, a comprehensive software, provided the final recommendation. Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual factors. In G. uralensis, the top three most stable reference genes across different tissues were R3HDM2, CAC and TUB, while CAC, CYP and ABCC2 were most suited for different developmental stages. CAC is the only candidate recommended for both biotic factors, which is reflected in the stability ranking for the spatio (tissue)-temporal (developmental stage) interactions (CAC, R3HDM2 and DNAJ). Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis. In summary, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential.

scholarly journals Influence of Angiopoietin Treatment with Hypoxia and Normoxia on Human Intervertebral Disc Progenitor Cell’s Proliferation, Metabolic Activity, and Phenotype

2021 ◽  
Vol 11 (15) ◽  
pp. 7144
Author(s):  
Muriel C. Bischof ◽  
Sonja Häckel ◽  
Andrea Oberli ◽  
Andreas S. Croft ◽  
Katharina A. C. Oswald ◽  
...  
Keyword(s):  
Gene Expression ◽  
Intervertebral Disc ◽  
Metabolic Activity ◽  
Cell Metabolism ◽  
Trauma Patients ◽  
Low Back ◽  
Relative Gene Expression ◽  
Progenitor Cell Population ◽  
Ivd Degeneration ◽  
Relative Gene

Increasing evidence implicates intervertebral disc (IVD) degeneration as a major contributor to low back pain. In addition to a series of pathogenic processes, degenerated IVDs become vascularized in contrast to healthy IVDs. In this context, angiopoietin (Ang) plays a crucial role and is involved in cytokine recruitment, and anabolic and catabolic reactions within the extracellular matrix (ECM). Over the last decade, a progenitor cell population has been described in the nucleus pulposus (NP) of the IVD to be positive for the Tie2 marker (also known as Ang-1 receptor). In this study, we investigated the influence of Ang-1 and Ang-2 on human NP cell (Tie2+, Tie2- or mixed) populations isolated from trauma patients during 7 days in normoxia (21% O2) or hypoxia (≤ 5% O2). At the end of the process, the proliferation and metabolic activity of the NP cells were analyzed. Additionally, the relative gene expression of NP-related markers was evaluated. NP cells showed a higher proliferation depending on the Ang treatment. Moreover, the study revealed higher NP cell metabolism when cultured in hypoxia. Additionally, the relative gene expression followed, with an increase linked to the oxygen level and Ang concentration. Our study comparing different NP cell populations may be the start of new approaches for the treatment of IVD degeneration.

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