A Scientific Note of Housekeeping Genes for the Primitively Eusocial bee Euglossa viridissima Friese (Apidae: Euglossini)

Studies on the expression of genes in different contexts are essential to our understanding of the functioning of organisms and their adaptations to the environment. Gene expression studies require steps of normalization, which are done using the stable expression pattern of reference genes. For many different eusocial bees reference genes have been discovered, but not for the primitively eusocial euglossine bees.We used available genomic resources of euglossine species and the gene information of Apis melliferato develop a set of reference genes for the primitive eusocial bee Euglossaviridissima. We tested nine genes in distinct developmental stages three different algorithms to infer the stability of gene expression. The Tata binding protein(Tbp) and 14-3-3epsilon were the most stable genes across all different stages. The strongest deviation in gene expression pattern occurred in pupae, which require a different set of genes for normalizing gene expression.

Download Full-text

Selection and Validation of Reference Genes for RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress

BioMed Research International ◽

10.1155/2021/4853632 ◽

2021 ◽

Vol 2021 ◽

pp. 1-12

Author(s):

Hao Xie ◽

Bo Li ◽

Yu Chang ◽

Xiaoyan Hou ◽

Yue Zhang ◽

...

Keyword(s):

Gene Expression ◽

Expression Pattern ◽

Reference Genes ◽

18S Rrna ◽

Spinacia Oleracea ◽

Expression Patterns ◽

Gene Expression Pattern ◽

Housekeeping Genes ◽

Qpcr Analysis ◽

The Stability

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an accurate and convenient method for mRNA quantification. Selection of optimal reference gene(s) is an important step in RT-qPCR experiments. However, the stability of housekeeping genes in spinach (Spinacia oleracea) under various abiotic stresses is unclear. Evaluating the stability of candidate genes and determining the optimal gene(s) for normalization of gene expression in spinach are necessary to investigate the gene expression patterns during development and stress response. In this study, ten housekeeping genes, 18S ribosomal RNA (18S rRNA), actin, ADP ribosylation factor (ARF), cytochrome c oxidase subunit 5C (COX), cyclophilin (CYP), elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (H3), 50S ribosomal protein L2 (RPL2), and tubulin alpha chain (TUBα) from spinach, were selected as candidates in roots, stems, leaves, flowers, and seedlings in response to high temperature, CdCl2, NaCl, NaHCO3, and Na2CO3 stresses. The expression of these genes was quantified by RT-qPCR and evaluated by NormFinder, BestKeeper, and geNorm. 18S rRNA, actin, ARF, COX, CYP, EF1α, GAPDH, H3, and RPL2 were detected as optimal reference genes for gene expression analysis of different organs and stress responses. The results were further confirmed by the expression pattern normalized with different reference genes of two heat-responsive genes. Here, we optimized the detection method of the gene expression pattern in spinach. Our results provide the optimal candidate reference genes which were crucial for RT-qPCR analysis.

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

Identification of reference genes for studies of quantitative gene expression in male and female quail tissues.

10.21203/rs.3.rs-355959/v1 ◽

2021 ◽

Author(s):

Maise dos Santos Macario ◽

C. S. Nascimento ◽

F. C. B. Sousa ◽

I. R. S. Oliveira ◽

A. P. D. Vesco ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Housekeeping Genes ◽

Thigh Muscle ◽

Male And Female ◽

Female Quail ◽

Expression Studies ◽

Cq Method ◽

Gene Expression Studies ◽

The Stability

Abstract This study aimed to evaluate stability and recommend reference genes for quantitative real-time PCR in different tissues from male and female broiler quails. The stability of 10 housekeeping genes (GAPDH, RPL5, MRPS27, MRPS30, TFRC, HMBS, EEF1, LDHA, B2M and UBC) was analyzed in heart, thigh muscle, brain and spleen, by means Bestkeeper, NormFinder, GeNorm softwares with ∆Cq method. The most stable housekeeping genes were MRPS30, TFRC and HMBS in heart; MRPS30, EEF1 and HMBS in thigh muscle; B2M, GAPDH and UBC in brain; and EEF1, LDHA and HMBS in spleen. It is recommended to be used as reference genes for gene expression studies of male and female quails.

Download Full-text

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

Scientific Reports ◽

10.1038/s41598-020-79298-8 ◽

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.

Download Full-text

Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR

Scientific Reports ◽

10.1038/s41598-019-49474-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Dorota M. Krzyżanowska ◽

Anna Supernat ◽

Tomasz Maciąg ◽

Marta Matuszewska ◽

Sylwia Jafra

Keyword(s):

Gene Expression ◽

Quorum Sensing ◽

Reference Genes ◽

Significance Threshold ◽

Expression Studies ◽

Reverse Transcription Quantitative Pcr ◽

Gene Expression Studies ◽

The Stability ◽

Selection Of

Abstract Reverse transcription quantitative PCR (RT-qPCR), a method of choice for quantification of gene expression changes, requires stably expressed reference genes for normalization of data. So far, no reference genes were established for the Alphaproteobacteria of the genus Ochrobactrum. Here, we determined reference genes for gene expression studies in O. quorumnocens A44. Strain A44 was cultured under 10 different conditions and the stability of expression of 11 candidate genes was evaluated using geNorm, NormFinder and BestKeeper. Most stably expressed genes were found to be rho, gyrB and rpoD. Our results can facilitate the choice of reference genes in the related Ochrobactrum strains. O. quorumnocens A44 is able to inactivate a broad spectrum of N-acyl homoserine lactones (AHLs) – the quorum sensing molecules of many Gram-negative bacteria. This activity is attributed to AiiO hydrolase, yet it remains unclear whether AHLs are the primary substrate of this enzyme. Using the established RT-qPCR setup, we found that the expression of the aiiO gene upon exposure to two AHLs, C6-HLS and 3OC12-HSL, does not change above the 1-fold significance threshold. The implications of this finding are discussed in the light of the role of quorum sensing-interfering enzymes in the host strains.

Download Full-text

Selecting and validating reference genes for quantitative real-time PCR in Plutella xylostella (L.)

Genome ◽

10.1139/gen-2017-0176 ◽

2018 ◽

Vol 61 (5) ◽

pp. 349-358 ◽

Cited By ~ 6

Author(s):

Yanchun You ◽

Miao Xie ◽

Liette Vasseur ◽

Minsheng You

Keyword(s):

Gene Expression ◽

Real Time ◽

Plutella Xylostella ◽

Real Time Pcr ◽

Reference Genes ◽

Developmental Stages ◽

Quantitative Real Time Pcr ◽

Experimental Conditions ◽

Expression Studies ◽

Gene Expression Studies

Gene expression analysis provides important clues regarding gene functions, and quantitative real-time PCR (qRT-PCR) is a widely used method in gene expression studies. Reference genes are essential for normalizing and accurately assessing gene expression. In the present study, 16 candidate reference genes (ACTB, CyPA, EF1-α, GAPDH, HSP90, NDPk, RPL13a, RPL18, RPL19, RPL32, RPL4, RPL8, RPS13, RPS4, α-TUB, and β-TUB) from Plutella xylostella were selected to evaluate gene expression stability across different experimental conditions using five statistical algorithms (geNorm, NormFinder, Delta Ct, BestKeeper, and RefFinder). The results suggest that different reference genes or combinations of reference genes are suitable for normalization in gene expression studies of P. xylostella according to the different developmental stages, strains, tissues, and insecticide treatments. Based on the given experimental sets, the most stable reference genes were RPS4 across different developmental stages, RPL8 across different strains and tissues, and EF1-α across different insecticide treatments. A comprehensive and systematic assessment of potential reference genes for gene expression normalization is essential for post-genomic functional research in P. xylostella, a notorious pest with worldwide distribution and a high capacity to adapt and develop resistance to insecticides.

Download Full-text

Selection and validation of reference genes for quantitative gene expression analyses in persimmon (Diospyros kaki Thunb.) using real-time quantitative PCR

Biologia Futura ◽

10.1556/019.70.2019.24 ◽

2019 ◽

Vol 70 (4) ◽

pp. 261-267

Author(s):

Gaigai Du ◽

Liyuan Wang ◽

Huawei Li ◽

Peng Sun ◽

Jianmin Fu ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Reference Genes ◽

Developmental Stages ◽

Suitable Reference Gene ◽

Diospyros Kaki ◽

Stable Gene ◽

Expression Studies ◽

Gene Expression Studies

Background and aims Persimmon (Diospyros kaki) is an economically important fruit tree species with complex flowering characteristics. To obtain accurate expression pattern analysis results, it is vital to select a reliable gene for the normalization of real-time quantitative polymerase chain reaction data. The aim of this study was to identify the optimal internal control gene among six candidate genes for gene expression analysis in different persimmon organs and developmental stages. Materials and methods This analysis was conducted using geNorm and NormFinder software to show differences in the stability of the six reference genes among tissues and floral developmental stages of the same plant. Results Although genes that exhibited moderate expression in NormFinder revealed slightly different expression stabilities than those obtained by geNorm, both sets of results showed that GAPDH was the best reference gene in different organs and floral buds at different developmental stages, whereas 18SrRNA was the least stable gene. Conclusions Based on the overall ranking, GAPDH is the most suitable reference gene and is highly recommended for gene expression studies in different organs and different developmental stages of persimmon. This study provides useful reference data for future gene expression studies and will contribute to improving the accuracy of gene expression results in persimmon.

Download Full-text

Evaluation of Appropriate Reference Genes For Investigating Gene Expression in Chlorops oryzae (Diptera: Chloropidae)

Journal of Economic Entomology ◽

10.1093/jee/toz142 ◽

2019 ◽

Vol 112 (5) ◽

pp. 2207-2214 ◽

Cited By ~ 1

Author(s):

Ping Tian ◽

Lin Qiu ◽

Ailin Zhou ◽

Guo Chen ◽

Hualiang He ◽

...

Keyword(s):

Gene Expression ◽

Ribosomal Protein ◽

Reference Genes ◽

Molecular Mechanisms ◽

Developmental Stages ◽

Quantitative Polymerase Chain Reaction ◽

Housekeeping Genes ◽

Future Research ◽

Economic Damage ◽

Physiological Processes

Abstract Reverse transcription quantitative polymerase chain reaction (PCR) has become an invaluable technique for analyzing gene expression in many insects. However, this approach requires the use of stable reference genes to normalize the data. Chlorops oryzae causes significant economic damage to rice crops throughout Asia. The lack of suitable reference genes has hindered research on the molecular mechanisms underlying many physiological processes of this species. In this study, we used quantitative real-time PCR to evaluate the expression of eight C. oryzae housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (βACT), beta-tubulin (βTUB), Delta Elongation factor-1 (EF1δ), ribosomal protein S11 (RPS11), RPS15, C-terminal-Binding Protein (CtBP), and ribosomal protein 49 (RP49) in different developmental stages and tissues in both larvae and adults. We analyzed the data with four different software packages: geNorm, NormFinder, BestKeeper, and RefFinder and compared the results obtained with each method. The results indicate that PRS15 and RP49 can be used as stable reference genes for quantifying gene expression in different developmental stages and larval tissues. GAPDH and βACT, which have been considered stable reference genes by previous studies, were the least stable of the candidate genes with respect to larval tissues. GAPDH was, however, the most stable reference gene for adult tissues. We verified the candidate reference genes identified and found that the expression levels of Cadherins (Cads) changed when different reference genes were used to normalize gene expression. This study provides a valuable foundation for future research on gene function, and investigating the molecular basis of physiological processes, in C. oryzae.

Download Full-text

Ring sideroblasts in AML are associated with adverse risk characteristics and have a distinct gene expression pattern

Blood Advances ◽

10.1182/bloodadvances.2019000518 ◽

2019 ◽

Vol 3 (20) ◽

pp. 3111-3122

Author(s):

Gerbrig Berger ◽

Mylene Gerritsen ◽

Guoqiang Yi ◽

Theresia N. Koorenhof-Scheele ◽

Leonie I. Kroeze ◽

...

Keyword(s):

Gene Expression ◽

Expression Pattern ◽

Gene Expression Pattern ◽

Erythroid Differentiation ◽

Tp53 Mutations ◽

Expression Studies ◽

Risk Characteristics ◽

Key Points ◽

Ring Sideroblasts ◽

Gene Expression Studies

Key Points Ring sideroblasts in AML are associated with complex karyotypes and TP53 mutations. Gene expression studies in CD34+ AML cells suggest an altered erythroid differentiation program in AML with ring sideroblasts.

Download Full-text

Evaluation of putative reference genes for quantitative real-time PCR normalization inLilium regaleduring development and under stress

PeerJ ◽

10.7717/peerj.1837 ◽

2016 ◽

Vol 4 ◽

pp. e1837 ◽

Cited By ~ 15

Author(s):

Qiang Liu ◽

Chi Wei ◽

Ming-Fang Zhang ◽

Gui-Xia Jia

Keyword(s):

Gene Expression ◽

Heat Stress ◽

Real Time ◽

Quantitative Pcr ◽

Biotic Stress ◽

Reference Genes ◽

Developmental Stages ◽

Real Time Quantitative Pcr ◽

The Stability ◽

Experimental Treatments

Normalization to reference genes is the most common method to avoid bias in real-time quantitative PCR (qPCR), which has been widely used for quantification of gene expression. Despite several studies on gene expression,Lilium, and particularlyL. regale, has not been fully investigated regarding the evaluation of reference genes suitable for normalization. In this study, nine putative reference genes, namely18S rRNA,ACT,BHLH,CLA,CYP,EF1,GAPDH,SANDandTIP41, were analyzed for accurate quantitative PCR normalization at different developmental stages and under different stress conditions, including biotic (Botrytis elliptica), drought, salinity, cold and heat stress. All these genes showed a wide variation in their Cq (quantification Cycle) values, and their stabilities were calculated by geNorm, NormFinder and BestKeeper. In a combination of the results from the three algorithms,BHLHwas superior to the other candidates when all the experimental treatments were analyzed together;CLAandEF1were also recommended by two of the three algorithms. As for specific conditions,EF1under various developmental stages,SANDunder biotic stress,CYP/GAPDHunder drought stress, andTIP41under salinity stress were generally considered suitable. All the algorithms agreed on the stability ofSANDandGAPDHunder cold stress, while onlyCYPwas selected under heat stress by all of them. Additionally, the selection of optimal reference genes under biotic stress was further verified by analyzing the expression level ofLrLOXin leaves inoculated withB. elliptica. Our study would be beneficial for future studies on gene expression and molecular breeding ofLilium.

Download Full-text