Identification of qPCR reference genes suitable for normalising gene expression in the developing mouse embryo

Background: Progression through mammalian embryogenesis involves many interacting cell types and multiple differentiating cell lineages. Quantitative polymerase chain reaction (qPCR) analysis of gene expression in the developing embryo is a valuable tool for deciphering these processes, but normalisation to stably-expressed reference genes is essential for such analyses. Gene expression patterns change globally and dramatically as embryonic development proceeds, rendering identification of consistently appropriate reference genes challenging. Methods: We have investigated expression stability in mouse embryos from mid to late gestation (E11.5–E18.5), both at the whole-embryo level, and within the head and forelimb specifically, using 15 candidate reference genes (ACTB, 18S, SDHA, GAPDH, HTATSF1, CDC40, RPL13A, CSNK2A2, AP3D1, HPRT1, CYC1, EIF4A, UBC, B2M and PAK1IP1), and four complementary algorithms (geNorm, Normfinder, Bestkeeper and deltaCt). Results: Unexpectedly, all methods suggest that many genes within our candidate panel are acceptable references, though AP3D1, RPL13A and PAK1IP1 are the strongest performing genes overall. HPRT1 and B2M are conversely poor choices, and show strong developmental regulation. We further show that normalisation using our three highest-scoring references can reveal subtle patterns of developmental expression even in genes ostensibly ranked as acceptably stable (CDC40, HTATSF1). Conclusion: AP3D1, RPL13A and PAK1IP1 represent universally suitable reference genes for expression studies in the E11.5-E18.5 mouse embryo.

Download Full-text

Identification of qPCR reference genes suitable for normalising gene expression in the developing mouse embryo

10.1101/2020.10.29.360461 ◽

2020 ◽

Author(s):

John C.W. Hildyard ◽

Dominic J. Wells ◽

Richard J. Piercy

Keyword(s):

Gene Expression ◽

Mouse Embryo ◽

Reference Genes ◽

Developmental Regulation ◽

Cell Types ◽

Tissue Level ◽

Late Gestation ◽

Developmental Expression ◽

Pcr Analysis ◽

Expression Change

AbstractMammalian embryogenesis is an intricate, tightly orchestrated process. Progression from zygote through somitogenesis and on to organogenesis and maturity involves many interacting cell types and multiple differentiating cell lineages. Quantitative PCR analysis of gene expression in the developing embryo is a valuable tool for deciphering these interactions and tracing lineages, but normalisation of qPCR data to stably expressed reference genes is essential. Patterns of gene expression change globally and dramatically as embryonic development proceeds, rendering identification of appropriate reference genes challenging at both the whole embryo- and individual tissue-level. We have investigated expression stability in mouse embryos from mid to late gestation (E11.5–E18.5), both at the whole-embryo level, and within more restricted tissue domains (head, developing forelimb), using 15 candidate reference genes (ACTB, 18S, SDHA, GAPDH, HTATSF1, CDC40, RPL13A, CSNK2A2, AP3D1, HPRT1, CYC1, EIF4A, UBC, B2M and PAK1IP1), and four complementary algorithms (geNorm, Normfinder, Bestkeeper and deltaCt). Unexpectedly, all methods suggest that many genes within our candidate panel are acceptable references, and despite disagreement over highest-scoring candidates, AP3D1, RPL14A and PAK1IP1 are the strongest performing genes overall. Conversely, HPRT1 and B2M are consistently poor choices: these genes show strong developmental regulation. We further show that use of AP3D1, RPL13A and PAK1IP1 can reveal subtle patterns of developmental expression even in genes ostensibly ranked as acceptable (CDC40, HTATSF1), and thus these three represent universally suitable reference genes for the mouse embryo.

Download Full-text

Reference gene selection for qRT-PCR analyses of luffa (Luffa cylindrica) plants under abiotic stress conditions

Scientific Reports ◽

10.1038/s41598-021-81524-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Min-dong Chen ◽

Bin Wang ◽

Yong-ping Li ◽

Mei-juan Zeng ◽

Jian-ting Liu ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Gene Selection ◽

Expression Patterns ◽

Suitable Reference Gene ◽

Expression Levels ◽

Qrt Pcr ◽

Suitable Reference ◽

Gene Expression Levels

AbstractSelecting suitable internal reference genes is an important prerequisite for the application of quantitative real-time PCR (qRT-PCR). However, no systematic studies have been conducted on reference genes in luffa. In this study, seven reference genes were selected, and their expression levels in luffa plants exposed to various simulated abiotic stresses [i.e., cold, drought, heat, salt, H2O2, and abscisic acid (ABA) treatments] were analyzed by qRT-PCR. The stability of the reference gene expression levels was validated using the geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The results indicated that EF-1α was the most stably expressed and suitable reference gene overall and for the heat, cold, and ABA treatments. Additionally, UBQ expression was stable following the salt treatment, whereas TUB was identified as a suitable reference gene for H2O2 and drought treatments. The reliability of the selected reference genes was verified by analyzing the expression of copper/zinc superoxide dismutase (Cu/Zn-SOD) gene in luffa. When the most unstable reference genes were used for data normalizations, the resulting expression patterns had obvious biases when compared with the expression patterns for the most ideal reference genes used alone or combined. These results will be conducive to more accurate quantification of gene expression levels in luffa.

Download Full-text

Screening of reference genes using real-time quantitative PCR for gene expression studies inNeoseiulus barkeriHughes (Acari: Phytoseiidae)

Bulletin of Entomological Research ◽

10.1017/s000748531800072x ◽

2018 ◽

Vol 109 (4) ◽

pp. 443-452 ◽

Cited By ~ 1

Author(s):

C. Wang ◽

J. Yang ◽

Q. Pan ◽

S. Yu ◽

R. Luo ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Genes ◽

Developmental Stages ◽

Quantitative Polymerase Chain Reaction ◽

Real Time Quantitative Pcr ◽

Expression Studies ◽

Gene Expression Studies ◽

The Stability ◽

Suitable Reference

AbstractA stable reference gene is a key prerequisite for accurate assessment of gene expression. At present, the real-time reverse transcriptase quantitative polymerase chain reaction has been widely used in the analysis of gene expression in a variety of organisms.Neoseiulus barkeriHughes (Acari: Phytoseiidae) is a major predator of mites on many important economically crops. Until now, however, there are no reports evaluating the stability of reference genes in this species. In view of this, we used GeNorm, NormFinder, BestKeeper, and RefFinder software tools to evaluate the expression stability of 11 candidate reference genes in developmental stages and under various abiotic stresses. According to our results, β-ACTandHsp40were the top two stable reference genes in developmental stages. TheHsp60andHsp90were the most stable reference genes in various acaricides stress. For alterations in temperature,Hsp40and α-TUBwere the most suitable reference genes. About UV stress,EF1α and α-TUBwere the best choice, and for the different prey stress, β-ACTand α-TUBwere best suited. In normal conditions, the β-ACT and α-TUB were the two of the highest stable reference genes to respond to all kinds of stresses. The current study provided a valuable foundation for the further analysis of gene expression inN. barkeri.

Download Full-text

Selection of Reference Genes for qPCR Analyses of Gene Expression in Ramie Leaves and Roots across Eleven Abiotic/Biotic Treatments

Scientific Reports ◽

10.1038/s41598-019-56640-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Yongting Yu ◽

Gang Zhang ◽

Yikun Chen ◽

Qingqing Bai ◽

Chunsheng Gao ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Natural Fiber ◽

Expression Patterns ◽

Functional Studies ◽

Expression Studies ◽

Leaves And Roots ◽

Gene Expression Studies ◽

Suitable Reference ◽

Common Plant

AbstractQuantitative real-time PCR (qPCR) is commonly used for deciphering gene functions. For effective qPCR analyses, suitable reference genes are needed for normalization. The objective of this study is to identify the appropriate reference gene(s) for qPCR analyses of the leaves and roots of ramie (Boehmeria nivea L.), an important natural fiber crop. To accomplish this goal, we investigated the expression patterns of eight common plant qPCR reference genes in ramie leaves and roots under five abiotic stresses, five hormonal treatments, and one biotic stress. The relative expression stabilities of the eight genes were evaluated using four common but different approaches: geNorm, NormFinder, BestKeeper, and RefFinder. Across the 11 tested conditions, ACT1 was the most stably expressed among the eight genes while GAPDH displayed the biggest variation. Overall, while variations in the suggested reference genes were found for different tissue x treatment combinations, our analyses revealed that together, genes ACT1, CYP2, and UBQ can provide robust references for gene expression studies of ramie leaves under most conditions, while genes EF-1α, TUB, and ACT1 can be used for similar studies of ramie roots. Our results should help future functional studies of the genes in ramie genome across tissues and environmental conditions.

Download Full-text

Identification of reference genes for gene expression studies among different developmental stages of murine hearts

BMC Developmental Biology ◽

10.1186/s12861-021-00244-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Jie Ren ◽

Ningning Zhang ◽

Xiangjie Li ◽

Xiaogang Sun ◽

Jiangping Song

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Heart Development ◽

Reference Genes ◽

Developmental Stages ◽

Expression Profiles ◽

Quantitative Polymerase Chain Reaction ◽

Expression Patterns ◽

Gene Expression Profiles ◽

Qpcr Data

Abstract Background Real-time quantitative polymerase chain reaction (RT-qPCR) is a widely-used standard assay for assessing gene expression. RT-qPCR data requires reference genes for normalization to make the results comparable. Therefore, the selected reference gene should be highly stable in its expression throughout the experimental datasets. So far, reports about the optimal set of reference genes in murine left ventricle (LV) across embryonic and postnatal stages are few. The objective of our research was to identify the appropriate reference genes in murine LV among different developmental stages. Methods We investigated the gene expression profiles of 21 widely used housekeeping genes in murine LV from 7 different developmental stages (almost throughout the whole period of the mouse lifespan). The stabilities of the potential reference genes were evaluated by five methods: GeNorm, NormFinder, BestKeeper, Delta-Ct and RefFinder. Results We proposed a set of reliable reference genes for normalization of RT-qPCR experimental data in different conditions. Furthermore, our results showed that 6 genes (18S, Hmbs, Ubc, Psmb4, Tfrc and Actb) are not recommended to be used as reference genes in murine LV development studies. The data also suggested that the Rplp0 gene might serve as an optimal reference gene in gene expression analysis. Conclusions Our study investigated the expression stability of the commonly used reference genes in process of LV development and maturation. We proposed a set of optimal reference genes that are suitable for accurate normalization of RT-qPCR data in specific conditions. Our findings may be helpful in future studies for investigating the gene expression patterns and mechanism of mammalian heart development.

Download Full-text

Selection and validation of reference genes for quantitative gene expression normalization in Taxus spp.

Scientific Reports ◽

10.1038/s41598-020-79213-1 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Kaikai Zhang ◽

Wei Fan ◽

Duanfen Chen ◽

Luyuan Jiang ◽

Yunfeng Li ◽

...

Keyword(s):

Gene Expression ◽

Cell Line ◽

Reference Genes ◽

Expression Patterns ◽

Future Research ◽

Taxus Chinensis ◽

Experimental Conditions ◽

Taxol Biosynthesis ◽

Qrt Pcr ◽

Suitable Reference

AbstractQuantitative real-time PCR (qRT-PCR) is commonly used to measure gene expression to further explore gene function, while suitable reference genes must be stably expressed under different experimental conditions to obtain accurate and reproducible data for relative quantification. Taxol or paclitaxel is an important anticancer compound mainly identified in Taxus spp. The molecular mechanism of the regulation of taxol biosynthesis is current research goal. However, in the case of Taxus spp., few reports were published on screening suitable reference genes as internal controls for qRT-PCR. Here, eight reference genes were selected as candidate reference genes for further study. Common statistical algorithms geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder were used to analyze the data from samples collected from a cell line of Taxus × media under various experimental conditions and from tissues of Taxus chinensis var. mairei. The expression patterns of TcMYC under salicylic acid treatment differed significantly, with the best and worst reference genes in the cell line. This study screened out suitable reference genes (GAPDH1 and SAND) under different treatments and tissues for the accurate and reliable normalization of the qRT-PCR expression data of Taxus spp. At the same time, this study will aid future research on taxol biosynthesis-related genes expression in Taxus spp., and can also be directly used to other related species.

Download Full-text

Stable Reference Genes for qPCR Analysis in BM-MSCs Undergoing Osteogenic Differentiation within 3D Hyaluronan-Based Hydrogels

International Journal of Molecular Sciences ◽

10.3390/ijms21239195 ◽

2020 ◽

Vol 21 (23) ◽

pp. 9195

Author(s):

Johannes Hasler ◽

Luan Phelipe Hatt ◽

Martin James Stoddart ◽

Angela Rita Armiento

Keyword(s):

Gene Expression ◽

Osteogenic Differentiation ◽

Reference Genes ◽

High Throughput Screening ◽

Quantitative Polymerase Chain Reaction ◽

Cell Phenotype ◽

Research Fields ◽

Qpcr Analysis ◽

The Stability ◽

Suitable Reference

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) enables the monitoring of changes in cell phenotype via the high-throughput screening of numerous genes. RT-qPCR is a fundamental approach in numerous research fields, including biomaterials, yet little attention has been given to the potential impact of 3D versus monolayer (2D) cell culture and to the requirement for a constant validation of the multiple steps of gene expression analysis. The aim of this study is to use high-quality RNA to identify the most suitable reference genes for RT-qPCR analysis during the osteogenic differentiation of human bone marrow mesenchymal stem/stromal cells (BM-MSCs). BM-MSCs are cultured under osteogenic conditions for 28 days in 2D or within hyaluronic acid hydrogels (3D). RNA is subject to quality controls and is then used to identify the most stable reference genes using geNorm, NormFinder, and the ∆Cq method. The effect of the reverse transcriptase is investigated, as well as the expression of osteogenic-related markers. This study shows marked differences in the stability of reference genes between 2D (RPLP0/GAPDH) and 3D (OAZ1/PPIA) culture, suggesting that it is critical to choose appropriate reference genes for 3D osteogenic cell cultures. Thus, a thorough validation under specific experimental settings is essential to obtain meaningful gene expression results.

Download Full-text

Reference Gene Selection for qRT-PCR Analyses of Luffa (Luffa cylindrica) Plants Under Abiotic Stress Conditions

10.21203/rs.3.rs-65316/v1 ◽

2020 ◽

Author(s):

mindong chen ◽

bin wang ◽

yongping li ◽

meijuan zeng ◽

jianting liu ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Abiotic Stresses ◽

Reference Genes ◽

Expression Patterns ◽

Cold Treatment ◽

Suitable Reference Gene ◽

Expression Levels ◽

Qrt Pcr ◽

Suitable Reference

Abstract Background: Quantitative real-time PCR (qRT-PCR) is one of the preferred methods for analyzing gene expression, and selecting suitable internal reference genes is an important prerequisite for the application of this technology. However, no systematic studies have been conducted on reference genes in luffa, resulting in limited investigations of luffa gene expression. Results: In this study, seven reference genes ( ACT , TUA , TUB , EF-1α , GAPDH , UBQ , and 18S ) were selected, and their expression levels in luffa plants exposed to various simulated abiotic stresses [i.e., cold, drought, heat, salt, H 2 O 2 , and abscisic acid (ABA) treatments] were analyzed by qRT-PCR. The stability of the reference gene expression levels was validated using the geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The results indicated that EF-1α was the most stably expressed and suitable reference gene overall and for the heat, cold, and ABA treatments. Additionally, UBQ expression was stable following the salt treatment, whereas TUB was identified as a suitable reference gene for H 2 O 2 and drought treatments. In contrast, GAPDH was revealed as an unsuitable reference gene overall and for the heat, salt, H 2 O 2 , ABA, and drought treatments. Regarding the cold treatment, TUA was identified as an unsuitable reference gene. The reliability of the selected reference genes was verified by analyzing the expression of copper/zinc superoxide dismutase ( Cu/Zn-SOD ) gene in luffa. When the most unstable reference genes were used for data normalizations, the resulting expression patterns had obvious biases when compared with the expression patterns for the most ideal reference genes used alone or combined. Conclusions: The study data were used to compile a list of suitable reference genes for qRT-PCR analyses of the gene expression in luffa plants exposed to abiotic stresses. This work may provide the basis for future qRT-PCR-based investigations of the transcription of important functional genes in luffa.

Download Full-text

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

10.21203/rs.3.rs-208040/v1 ◽

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.

Download Full-text