κMicroarray analysis of relative gene expression stability for selection of internal reference genes in the rhesus macaque brain

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.

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Expression stability and selection of optimal reference genes for gene expression normalization in early life stage rainbow trout exposed to cadmium and copper

Aquatic Toxicology ◽

10.1016/j.aquatox.2017.07.009 ◽

2017 ◽

Vol 190 ◽

pp. 217-227 ◽

Cited By ~ 11

Author(s):

Kamran Shekh ◽

Song Tang ◽

Som Niyogi ◽

Markus Hecker

Keyword(s):

Gene Expression ◽

Rainbow Trout ◽

Reference Genes ◽

Early Life ◽

Life Stage ◽

Early Life Stage ◽

Expression Stability ◽

Selection Of

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

10.21203/rs.3.rs-507940/v1 ◽

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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Gene expression stability of candidate reference genes under different culture conditions for quantitative PCR in the raphidophyte Chattonella marina

Phycologia ◽

10.1080/00318884.2020.1816041 ◽

2020 ◽

Vol 59 (6) ◽

pp. 556-565

Author(s):

Koki Mukai ◽

Yohei Shimasaki ◽

Xuchun Qiu ◽

Yoko Kato-Unoki ◽

Kun Chen ◽

...

Keyword(s):

Gene Expression ◽

Quantitative Pcr ◽

Reference Genes ◽

Culture Conditions ◽

Expression Stability ◽

Chattonella Marina ◽

Gene Expression Stability

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Selection of Suitable Reference Genes Based on Transcriptomic Data in Ginkgo biloba under Different Experimental Conditions

Forests ◽

10.3390/f11111217 ◽

2020 ◽

Vol 11 (11) ◽

pp. 1217

Author(s):

Tingting Zhou ◽

Xiaoming Yang ◽

Fangfang Fu ◽

Guibin Wang ◽

Fuliang Cao

Keyword(s):

Gene Expression ◽

Ginkgo Biloba ◽

Reference Genes ◽

Developmental Stages ◽

Deciduous Tree ◽

Internal Reference ◽

Experimental Conditions ◽

Transcriptomic Data ◽

Suitable Reference ◽

Selection Of

Ginkgo biloba, a deciduous tree species in the Ginkgo family, has a long history of cultivation in China and is widely used in garden landscapes, medicine, food, and health products. However, few reports have focused on the systematic selection of optimal reference genes based on transcriptomic data in G. biloba. The purpose of our research was to select an internal reference gene suitable for different experimental conditions from thirteen candidate reference genes by the delta cycle threshold (ΔCt) method, geNorm, BestKeeper, NormFinder, and RefFinder programs. The reference genes were used for gene expression analyses of Ginkgo biloba. These results showed that elongation factor 1(EF1) and ubiquitin (UBI) were the best choices for samples of different ginkgo genotypes. The expression of UBI and HAS28 presented the most stable at different developmental stages of ginkgo, and EIF3I and RPII were considered as suitable reference genes in different tissues of ginkgo. For methyl jasmonate (MeJA) treatment, ACA and ACT were identified as the optimal reference genes. For cold stress treatment, RPII and EIF4E were chosen for the gene expression normalizations. HAS28 and GAPDH presented the most stable expression for the heat treatment. To validate the above results, a chalcone synthase gene (GbCHS) in ginkgo was amplified by quantitative real-time polymerase chain reaction (qRT-PCR). Our results provide different suitable reference genes for further gene expression studies in ginkgo.

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

Reproduction Fertility and Development ◽

10.1071/rd14089 ◽

2016 ◽

Vol 28 (6) ◽

pp. 795 ◽

Cited By ~ 5

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.

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Selection of Reference Genes for Transcription Studies on Adventitious Root Induction in Olive (Olea Europaea L.) Microshoots Considering Co-expression and Average Transcriptional Stability

10.21203/rs.3.rs-60001/v1 ◽

2020 ◽

Author(s):

Carlos Noceda ◽

Augusto Peixe ◽

Birgit Arnholdt-Schmitt

Keyword(s):

Gene Expression ◽

Gene Expression Data ◽

Reference Genes ◽

Ad Hoc ◽

Root Induction ◽

Expression Data ◽

Internal Reference ◽

Experimental Conditions ◽

Selection Of

Abstract BackgroungSelection of reference genes (RGs) for normalization of PCR-gene expression data includes two crucial steps: determination of the among-sample transcriptionally more stable genes and subsequent choosing of the most suitable genes as internal controls. Both steps can be carried-out through generally accepted strategies each having different strengths and weaknesses. The present study proposes to reinforce normalization of gene expression data by integrating and adding analytical revision at critical steps of those accepted procedures. Especially crucial is to counterbalance a higher representative number of RGs with a correspondent increase in their average transcriptional instability or a generalised co-expression trend among the samples. This methodological study used in vitro olive adventitious rooting as an experimental system, since the underlying morphogenetic process -wich is common to diverse species- is still not completely understood.ResultsFirstly, RG candidates were ranked according to transcriptional stability following a simple statistical method that reduces biasing effects of concomitant, systematic biological variations associated to experimental conditions, such as the variations caused by gene co-regulation. Those types of systematic co-variation are unconsidered by several popular ad hoc informatics programmes. To select the adequate genes among those already ranked, an algorithm of one of the ad hoc informatics programmes (GeNorm) was adapted to allow partial automatization of RG selection for any strategy of transcriptional-gene stability ordering. In order to delve into the resulting possible RG sets suitability for inter-assay comparisons and technical-error compensation, separate statistics were formulated. The achieved results were compared with those obtained by standard stability ranking methods. Finally, a double evaluation was performed to accurately contrast two choice RG sets. The whole strategy was applied to a panel considering several independent factors, but the suitability of the obtained putative RG sets was tested for cases restricted to fewer variables. H2B, OUB and ACT are valid for normalization in transcriptional studies on olive microshoot rooting when comparing treatments, time points and assays.ConclusionsThe set of genes identified as internal reference is now available for wider expression studies on any target gene in similar biological systems. The overall methodology aims to constitute a guide for general application.

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

PeerJ ◽

10.7717/peerj.7181 ◽

2019 ◽

Vol 7 ◽

pp. e7181 ◽

Cited By ~ 2

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.

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The Selection of Reference Genes for Quantitative Real-Time PCR in the Ashidan Yak Mammary Gland During Lactation and Dry Period

Animals ◽

10.3390/ani9110943 ◽

2019 ◽

Vol 9 (11) ◽

pp. 943 ◽

Cited By ~ 4

Author(s):

Xiaoyun Wu ◽

Xuelan Zhou ◽

Xuezhi Ding ◽

Min Chu ◽

Chunnian Liang ◽

...

Keyword(s):

Gene Expression ◽

Mammary Gland ◽

Real Time ◽

Milk Yield ◽

Reference Genes ◽

Quantitative Polymerase Chain Reaction ◽

Expression Stability ◽

Dry Period ◽

Relative Expression ◽

Selection Of

Investigating the critical genes related to milk synthesis is essential for the improvement of the milk yield of the yak. Real-time quantitative polymerase chain reaction (RT-qPCR) is a reliable and widely used method to measure and evaluate gene expression levels. Selection of suitable reference genes is mandatory to acquire accurate normalization of gene expression results from RT-qPCR. To select the most stable reference genes for reliable normalization of mRNA expression by RT-qPCR in the mammary gland of the Ashidan yak, we selected 16 candidate reference genes and analyzed their expression stability at different physiological stages (lactation and dry period). The expression stability of the candidate reference genes was assessed using geNorm, NormFinder, BestKeeper, Delta Ct, and RefFinder methods. The results showed that the hydroxymethylbilane synthase gene (HMBS) and the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide gene (YWHAZ) were the most stable genes across all treatment samples. The reliability of selected reference genes was validated by normalizing relative expression of the lactation-related 60S ribosomal protein L35 gene (RPL35). The relative expression of RPL35 varied considerably according to the different reference genes. This work provides valuable information to further promote research in the molecular mechanisms involved in lactation and mammary gland development and provides a foundation for the improvement of the milk yield and quality of the Ashidan yak.

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