scholarly journals Identification of reliable reference genes for gene expression studies in maternal reproductive tissues and fetal tissues of pregnant cows

2020 ◽  
Author(s):  
Lei Cheng ◽  
Jie Yu ◽  
Xiuzhong Hu ◽  
Min Xiang ◽  
Yu Xia ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Target Genes ◽  
Quantitative Polymerase Chain Reaction ◽  
Related Factor ◽  
Expression Stability ◽  
Relative Expression ◽  
Reproductive Tissues ◽  
Fetal Tissues ◽  
Selection Of

Abstract Background: The relationship between the conceptus and the maternal uterine environment is crucial for the successful establishment and maintenance of pregnancy in cattle. Gene expression analysis of the conceptus and maternal reproductive tissues is a favorable method to assess the embryonic maternal interaction. The reliability of the commonly used method reverse transcription-quantitative polymerase chain reaction (RT-qPCR) depends on proper normalization to stable reference genes (RGs). The objective of this study was to determine the expression stability of ten potential RGs (SUZ12, CNOT11, ACTB, RPL19, RPS9, GAPDH, TBP, HPRT1, SDHA and PPIA) in maternal reproductive tissues and fetal tissues, and to analyze the effect of RG selection on the calculation of the relative expression of target genes. Results: The expression stability of ten potential RGs was analyzed in eight different tissues (caruncular endometrium, intercaruncular endometrium, corpus luteum, ovary, oviduct, mammary gland, embryonic disc and trophoblast) from three pregnant dairy cows. Three programs—GeNorm, NormFinder and Bestkeeper—were used to identify the best RGs. According to all three programs, the most stable RG was CNOT11, whereas the least stable RGs were GAPDH and HPRT1. GeNorm analysis showed that a combination of five RGs (SDHA, PPIA, CNOT11, RPS9 and RPL19) was necessary for appropriate data normalization. However, NormFinder analysis indicated that the combination of CNOT11 and PPIA was the most suitable. When target genes were normalized to these RGs, the relative expression of the Radical S-adenosyl methionine domain containing 2 gene was not affected by the choice of RGs, whereas a large difference was observed in the expression profile of the Nuclear erythroid2-related factor 2 gene between the most stable RGs and least stable RGs. Conclusions: The results indicate that careful selection of RGs is crucial under different conditions, especially for target genes with relatively small fold changes. Furthermore, the results provide useful information for the selection of RGs for evaluating genes affecting bovine reproduction.

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

Animals ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 943 ◽  
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.

Identification of reference genes for gene expression studies during seed germination and seedling establishment in Ricinus communis L.

2014 ◽  
Vol 24 (4) ◽  
pp. 341-352 ◽  
Author(s):  
Paulo R. Ribeiro ◽  
Bas J. W. Dekkers ◽  
Luzimar G. Fernandez ◽  
Renato D. de Castro ◽  
Wilco Ligterink ◽  
...  
Keyword(s):  
Gene Expression ◽  
Seed Germination ◽  
Reference Genes ◽  
Target Genes ◽  
Ricinus Communis ◽  
Quantitative Polymerase Chain Reaction ◽  
Optimal Combination ◽  
Expression Levels ◽  
Gene Expression Studies ◽  
Gene Expression Levels

AbstractReverse transcription-quantitative polymerase chain reaction (RT-qPCR) is an important technology to analyse gene expression levels during plant development or in response to different treatments. An important requirement to measure gene expression levels accurately is a properly validated set of reference genes. In this context, we analysed the potential use of 17 candidate reference genes across a diverse set of samples, including several tissues, different stages and environmental conditions, encompassing seed germination and seedling growth in Ricinus communis L. These genes were tested by RT-qPCR and ranked according to the stability of their expression using two different approaches: GeNorm and NormFinder. GeNorm and Normfinder indicated that ACT, POB and PP2AA1 comprise the optimal combination for normalization of gene expression data in inter-tissue (heterogeneous sample panel) studies. We also describe the optimal combination of reference genes for a subset of root, endosperm and cotyledon samples. In general, the most stable genes suggested by GeNorm are very consistent with those indicated by NormFinder, which highlights the strength of the selection of reference genes in our study. We also validated the selected reference genes by normalizing the expression levels of three target genes involved in energy metabolism with the reference genes suggested by GeNorm and NormFinder. The approach used in this study to identify stably expressed genes, and thus potential reference genes, was applied successfully for R. communis and it provides important guidelines for RT-qPCR studies in seeds and seedlings for other species (especially in those cases where extensive microarray data are not available).

scholarly journals Identification and Validation of Reference Genes for Gene Expression Analysis in Different Development Stages of Amylostereum areolatum

2022 ◽  
Vol 12 ◽  
Author(s):  
Ningning Fu ◽  
Jiaxing Li ◽  
Ming Wang ◽  
Lili Ren ◽  
Shixiang Zong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Growth And Development ◽  
Target Genes ◽  
Quantitative Polymerase Chain Reaction ◽  
Growth Stages ◽  
Experimental Conditions ◽  
Development Stages ◽  
Symbiotic Fungus ◽  
The Stability

A strict relationship exists between the Sirex noctilio and the Amylostereum areolatum, which is carried and spread by its partner. The growth and development of this symbiotic fungus is key to complete the life history of the Sirex woodwasp. Real-time quantitative polymerase chain reaction (RT-qPCR) is used to measure gene expression in samples of A. areolatum at different growth stages and explore the key genes and pathways involved in the growth and development of this symbiotic fungus. To obtain accurate RT-qPCR data, target genes need to be normalized by reference genes that are stably expressed under specific experimental conditions. In our study, the stability of 10 candidate reference genes in symbiotic fungal samples at different growth and development stages was evaluated using geNorm, NormFinder, BestKeeper, delta Ct methods, and RefFinder. Meanwhile, laccase1 was used to validate the stability of the selected reference gene. Under the experimental conditions of this study, p450, CYP, and γ-TUB were identified as suitable reference genes. This work is the first to systematically evaluate the reference genes for RT-qPCR results normalization during the growth of this symbiotic fungus, which lays a foundation for further gene expression experiments and understanding the symbiotic relationship and mechanism between S. noctilio and A. areolatum.

scholarly journals Identification of reliable reference genes for gene expression studies in maternal reproductive tissues and fetal tissues of pregnant cows

2020 ◽  
Author(s):  
Lei Cheng ◽  
Jie Yu ◽  
Xiuzhong Hu ◽  
Min Xiang ◽  
Yu Xia ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Reproductive Tissues ◽  
Fetal Tissues ◽  
Expression Studies ◽  
Gene Expression Studies

Abstract The authors have withdrawn this preprint due to author disagreement.

scholarly journals There is no magic bullet: the importance of testing reference gene stability in RT-qPCR experiments across multiple closely related species

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9618
Author(s):  
Bert Foquet ◽  
Hojun Song
Keyword(s):  
Gene Expression ◽  
Related Species ◽  
Reference Genes ◽  
Target Genes ◽  
Quantitative Polymerase Chain Reaction ◽  
Magic Bullet ◽  
Experimental Conditions ◽  
Closely Related Species ◽  
Expression Levels ◽  
Gene Stability

Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR) is the current gold standard tool for the study of gene expression. This technique is highly dependent on the validation of reference genes, which exhibit stable expression levels among experimental conditions. Often, reference genes are assumed to be stable a priori without a rigorous test of gene stability. However, such an oversight can easily lead to misinterpreting expression levels of target genes if the references genes are in fact not stable across experimental conditions. Even though most gene expression studies focus on just one species, comparative studies of gene expression among closely related species can be very informative from an evolutionary perspective. In our study, we have attempted to find stable reference genes for four closely related species of grasshoppers (Orthoptera: Acrididae) that together exhibit a spectrum of density-dependent phenotypic plasticity. Gene stability was assessed for eight reference genes in two tissues, two experimental conditions and all four species. We observed clear differences in the stability ranking of these reference genes, both between tissues and between species. Additionally, the choice of reference genes clearly influenced the results of a gene expression experiment. We offer suggestions for the use of reference genes in further studies using these four species, which should be taken as a cautionary tale for future studies involving RT-qPCR in a comparative framework.

scholarly journals Genome-Wide Identification of Reference Genes for Reverse-Transcription Quantitative PCR in Goat Rumen

Animals ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 3137
Author(s):  
Juan Zhao ◽  
Cheng Wang ◽  
Lin Zhang ◽  
Aiai Lei ◽  
Linjie Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quantitative Pcr ◽  
Reference Genes ◽  
Target Genes ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Expression Stability ◽  
Transport Pathway ◽  
Metabolic Role ◽  
Suitable Reference

As the largest chamber of the ruminant stomach, the rumen not only serves as the principal absorptive surface and nutrient transport pathway from the lumen into the animal, but also plays an important short-chain fatty acid (SCFA) metabolic role in addition to protective functions. Accurate characterization of the gene expression profiles of genes of interest is essential to the exploration of the intrinsic regulatory mechanisms of rumen development in goats. Thus, the selection of suitable reference genes (RGs) is an important prerequisite for real-time quantitative PCR (RT-qPCR). In the present study, 16 candidate RGs were identified from our previous transcriptome sequencing of caprine rumen tissues. The quantitative expressions of the candidate RGs were measured using the RT-qPCR method, and the expression stability of the RGs was assessed using the geNorm, NormFinder, and BestKeeper programs. GeNorm analysis showed that the M values were less than 0.5 for all the RGs except GAPT4, indicating that they were stably expressed in the rumen tissues throughout development. RPS4X and RPS6 were the two most stable RGs. Furthermore, the expressions of two randomly selected target genes (IGF1 and TOP2A), normalized by the selected most stable RGs (RPS4X and RPS6), were consistent with the results of RNA sequencing, while the use of GAPDH and ACTB as RGs resulted in altered profiles. Overall, RPS4X and RPS6 showed the highest expression stability and the lowest coefficients of variation, and could be used as the optimal reference combination for quantifying gene expression in rumen tissues via RT-qPCR analysis.

scholarly journals Selection of Suitable Reference Genes for RT-qPCR Gene Expression Analysis in Siberian Wild Rye (Elymus sibiricus) under Different Experimental Conditions

Genes ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 451 ◽  
Author(s):  
Junchao Zhang ◽  
Wengang Xie ◽  
Xinxuan Yu ◽  
Zongyu Zhang ◽  
Yongqiang Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Expression Stability ◽  
Forage Production ◽  
Experimental Conditions ◽  
Elymus Sibiricus ◽  
Suitable Reference ◽  
Selection Of

Elymus sibiricus, which is a perennial and self-pollinated grass, is the typical species of the genus Elymus, which plays an important role in forage production and ecological restoration. No reports have, so far, systematically described the selection of optimal reference genes for reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analysis in E. sibiricus. The goals of this study were to evaluate the expression stability of 13 candidate reference genes in different experimental conditions, and to determine the appropriate reference genes for gene expression analysis in E. sibiricus. Five methods including Delta Ct (ΔCt), BestKeeper, NormFinder, geNorm, and RefFinder were used to assess the expression stability of 13 potential reference genes. The results of the RefFinder analysis showed that TBP2 and HIS3 were the most stable reference genes in different genotypes. TUA2 and PP2A had the most stable expression in different developmental stages. TBP2 and PP2A were suitable reference genes in different tissues. Under salt stress, ACT2 and TBP2 were identified as the most stable reference genes. ACT2 and TUA2 showed the most stability under heat stress. For cold stress, PP2A and ACT2 presented the highest degree of expression stability. DNAJ and U2AF were considered as the most stable reference genes under osmotic stress. The optimal reference genes were selected to investigate the expression pattern of target gene CSLE6 in different conditions. This study provides suitable reference genes for further gene expression analysis using RT-qPCR in E. sibiricus.

scholarly journals Identification of appropriate reference genes for gene expression studies in mice left ventricles from different developmental stages

2021 ◽  
Author(s):  
Jie Ren ◽  
Ningning Zhang ◽  
Xiangjie Li ◽  
Xiaogang Sun ◽  
Jiangping Song
Keyword(s):  
Gene Expression ◽  
Heart Development ◽  
Reference Genes ◽  
Developmental Stages ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Patterns ◽  
Housekeeping Genes ◽  
Good Choice ◽  
Expression Stability ◽  
Gene Expressions

Abstract Aims: Real-time quantitative polymerase chain reaction (RT-qPCR) is the standard assay used for revealing the gene expression characteristics. However, the RT-qPCR studies all need reference genes for normalization to make the results comparable, which should hold a high expression stability during all experimental datasets. So far, there was no optimal set of reference genes identified in mice left ventricles (LV) across embryonic and postnatal stages. The objective of our research was to identify the appropriate reference genes in mice LV from different developmental stages.Methods and Results: we investigated the gene expressions of common 21 candidate housekeeping genes in mice LV from 7 different developmental stages, almost throughout the whole period of the mouse lifespan. The expression of some candidate reference genes, such as 18S and Actb, apparently fluctuated. The stability of potential reference genes was evaluated by a number of methods, such as GeNorm, NormFinder, BestKeeper, Delta-Ct and RefFinder method. we identified a set of optimal reference genes that can be reliably used for normalization of RT-qPCR experiments in different developmental stages of mice LV. Our results showed that following genes should not be used as reference genes in mice LV development studies: 18S, Hmbs, Ubc, Psmb4, Tfrc and Actb. And the Rplp0 appeared to represent a good choice. Conclusions: Our study provides the expression stability of the commonly used reference genes in process of LV development and maturation. We also identified a set of optimal reference genes under different conditions. Our findings may be helpful in future studies to investigate the gene expression patterns and mechanism of mammalian heart development.

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