Selection of candidate reference genes for RT-qPCR analysis in Argulus siamensis and their validation through screening of drugs and drug targets

AbstractArgulus spp. are economically important fish ectoparasites. The development of antiparasitic drugs is thus important and real time PCR is an indispensable tool in drug development. The analytical potential of RT-PCR depends upon accurate normalisation by the use of stable reference genes. Here, we identified stable reference genes of Argulus siamensis for validation of efficacy of drugs and drug targets. Seven candidate genes were evaluated by evaluating their expression under different states of Argulus using the RefFinder tool. The four algorithms together generated a comprehensive ranking with elongation factor-1 alpha (EF-1α) being the most stable and 18S ribosomal protein (18S) the least stable gene. Taking EF-1α and 18S genes as references, the effectiveness of six anti-parasitic compounds against Argulus was evaluated by studying their effect on the expression pattern of few ion channel genes; this was to understand their mode of action, besides validating the reference genes. EF-1α was found to be the most stable gene in the validation. Collectively, this study is the first report to validate the optimal reference genes of A. siamensis for normalisation, and the potential of the ion channel genes for evaluating effective drug targets in parasite control.

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Evaluation And Validation of Reliable Reference Genes For Quantitative Real-Time PCR Analysis of The Gene Expression In Macadamia Integrifolia

10.21203/rs.3.rs-645521/v1 ◽

2021 ◽

Author(s):

Qian Yang ◽

Ziping Yang ◽

Yali Zhou ◽

Hui Zeng ◽

Minghong Zou ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Geometric Mean ◽

Elongation Factor ◽

Stable Gene ◽

Expression Stability ◽

Tissue Samples ◽

Qrt Pcr ◽

Macadamia Integrifolia ◽

Elongation Factor 1

Abstract Background: Macadamia integrifolia, a new economically important crop, the kernel oil is rich in bioactive compound and monounsaturated fatty acid. Gene expression analysis of qRT-PCR is beneficial to understand the complex regulatory networks of macadamia.Results: In this study, the expression stability of 11 traditional housekeeping genes including α-tubulin (TUBa), β–tubulin (TUBb), malate dehydrogenase (MDH), 18S ribosome RNA (18S), glyceraldehyde-3- phosphate dehydrogenase (GAPDH), α-elongation factor 1 (EF1a), β- elongation factor 1 (EF1b), ubiquitin (UBQ), ubiquitin-conjugating enzyme (UBC), cyclophilin (CYP) and actin (ACT) were accessed by qRT-PCR in macadamia seedlings under different experimental conditions and tissues. The expression stability of the 11 reference genes was evaluated by the online tool RefFinder, which include ΔCt, geNorm, NormFinder, BestKeeper four commonly software, and then determinated a comprehensive expression stability ranking by integrating above four ranking results based on the geometric mean. Our results show that ACT was the best stable genes for all samples, cold stress, NaCl sress, PEG stress, ABA treatment, MeJA treatment, stem and leaf tissue samples; EF1b is the most stable gene in GA treatment and heat stress samples; UBC and CYP were respectively ranked top in ethylene treatment and root tissue samples. Finally, the reliability of these results was further validated with a target gene SAD by qRT-PCR. Conclusions: In summary, this study evaluated and validated the suitable reference genes for qRT-PCR under different experiment treatment and tissues, and will be useful for further gene expression studies on the molecular mechanisms in Macadamia.

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Selection of reference genes for gene expression analysis in Liriodendron hybrids’ somatic embryogenesis and germinative tissues

Scientific Reports ◽

10.1038/s41598-021-84518-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tingting Li ◽

Weigao Yuan ◽

Shuai Qiu ◽

Jisen Shi

Keyword(s):

Gene Expression ◽

Somatic Embryogenesis ◽

Reference Genes ◽

Gene Selection ◽

Elongation Factor ◽

Reverse Transcription Pcr ◽

Expression Of Genes ◽

Suitable Reference ◽

Functional Analyses ◽

Elongation Factor 1

AbstractThe differential expression of genes is crucial for plant somatic embryogenesis (SE), and the accurate quantification of gene expression levels relies on choosing appropriate reference genes. To select the most suitable reference genes for SE studies, 10 commonly used reference genes were examined in synchronized somatic embryogenic and subsequent germinative cultures of Liriodendron hybrids by using quantitative real-time reverse transcription PCR. Four popular normalization algorithms: geNorm, NormFinder, Bestkeeper and Delta-Ct were used to select and validate the suitable reference genes. The results showed that elongation factor 1-gamma, histone H1 linker protein, glyceraldehyde-3-phosphate dehydrogenase and α-tubulin were suitable for SE tissues, while elongation factor 1-gamma and actin were best for the germinative organ tissues. Our work will benefit future studies of gene expression and functional analyses of SE in Liriodendron hybrids. It is also serves as a guide of reference gene selection in early embryonic gene expression analyses for other woody plant species.

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Tissue-specific evaluation of suitable reference genes for RT-qPCR in the pond snail, Lymnaea stagnalis

10.7287/peerj.preprints.27695 ◽

2019 ◽

Author(s):

Alexander P Young ◽

Carmen F Landry ◽

Daniel J Jackson ◽

Russell C Wyeth

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Lymnaea Stagnalis ◽

Elongation Factor ◽

Pond Snail ◽

Tissue Samples ◽

Systematic Assessment ◽

Wide Range ◽

The Stability ◽

Suitable Reference

Reverse transcription quantitative PCR (RT-qPCR) is a robust technique for the quantification and comparison of gene expression across multiple tissues. To obtain reliable results, one or more reference genes must be employed to normalize expression measurements among treatments or tissue samples. Candidate reference genes must be validated to ensure that they are stable prior to use in qPCR experiments. The pond snail (Lymnaea stagnalis) is a common research organism, particularly in the areas of learning and memory, and is an emerging target for qPCR experimentation. However, no systematic assessment of reference genes has been performed in this animal. Therefore, the aim of our research was to identify stable reference genes to normalize gene expression data from a variety of tissues in L. stagnalis. We evaluated a panel of seven reference genes across six different tissues in L. stagnalis with RT-qPCR. The genes included: elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (ACTB), beta-tubulin (TUBB), ubiquitin (UBI), prenylated rab acceptor protein 1 (Rapac1), and a voltage gated potassium channel (VGKC). These genes exhibited a wide range of expression levels among tissues. The stability of each of the genes was consistent when measured by any of the standard stability assessment algorithms: geNorm, NormFinder, BestKeeper and RefFinder. Our data indicate that GAPDH and EF1α are highly stable in the tissues that we examined (central nervous system, tentacles, lips, penis, foot, mantle) as well as in pooled analyses. We do not recommend VGKC for use in RT-qPCR experiments due to its relatively low expression stability. Our results were generally congruent with those obtained from similar studies in other molluscs. Given that a minimum of two reference genes are recommended for data normalization, we suggest GAPDH and EF1α are a strong option for multi-tissue analyses of RT-qPCR data in Lymnaea stagnalis.

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Validation of Internal Control Genes for Quantitative Real-Time PCR Gene Expression Analysis in Morchella

Molecules ◽

10.3390/molecules23092331 ◽

2018 ◽

Vol 23 (9) ◽

pp. 2331 ◽

Cited By ~ 10

Author(s):

Qianqian Zhang ◽

Wei Liu ◽

Yingli Cai ◽

A-Feng Lan ◽

Yinbing Bian

Keyword(s):

Gene Expression ◽

Candidate Genes ◽

Expression Analysis ◽

Internal Control ◽

Reference Genes ◽

Gene Expression Analysis ◽

Stable Gene ◽

Experimental Conditions ◽

The Stability ◽

Vacuolar Protein

The reliability of qRT-PCR results depend on the stability of reference genes used for normalization, suggesting the necessity of identification of reference genes before gene expression analysis. Morels are edible mushrooms well-known across the world and highly prized by many culinary kitchens. Here, several candidate genes were selected and designed according to the Morchella importuna transcriptome data. The stability of the candidate genes was evaluated with geNorm and NormFinder under three different experimental conditions, and several genes with excellent stability were selected. The extensive adaptability of the selected genes was tested in ten Morchella species. Results from the three experimental conditions revealed that ACT1 and INTF7 were the most prominent genes in Morchella, CYC3 was the most stable gene in different development stages, INTF4/AEF3 were the top-ranked genes across carbon sources, while INTF3/CYC3 pair showed the robust stability for temperature stress treatment. We suggest using ACT1, AEF3, CYC3, INTF3, INTF4 and INTF7 as reference genes for gene expression analysis studies for any of the 10 Morchella strains tested in this study. The stability and practicality of the gene, vacuolar protein sorting (INTF3), vacuolar ATP synthase (INTF4) and14-3-3 protein (INTF7) involving the basic biological processes were validated for the first time as the candidate reference genes for quantitative PCR. Furthermore, the stability of the reference genes was found to vary under the three different experimental conditions, indicating the importance of identifying specific reference genes for particular conditions.

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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.

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The Selection of Reliable Reference Genes for RT-qPCR Analysis of Anisakis simplex Sensu Stricto Gene Expression from Different Developmental Stages

Acta Parasitologica ◽

10.2478/s11686-020-00220-3 ◽

2020 ◽

Vol 65 (4) ◽

pp. 837-842 ◽

Cited By ~ 1

Author(s):

Elżbieta Łopieńska-Biernat ◽

Robert Stryiński ◽

Łukasz Paukszto ◽

Jan Paweł Jastrzębski ◽

Karol Makowczenko

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Parasitic Nematode ◽

Developmental Stages ◽

Receptor Gene ◽

Elongation Factor ◽

Sensu Stricto ◽

Nuclear Hormone Receptor ◽

Anisakis Simplex ◽

Complex Life Cycle

Abstract Background Anisakis simplex s. s. is a parasitic nematode with a complex life cycle in which humans can become accidental hosts by consuming raw or not fully cooked fish containing L3 larvae. The growing popularity of raw fish dishes has contributed to an increase in the incidence of anisakiasis, which has spurred scientific efforts to develop new methods for diagnosing and treating the disease and also to investigate the gene expression at different developmental stages of this parasite. The identification of reference genes suitable for the normalization of RT-qPCR data has not been studied with respect to A. simplex s. s. Methods In the present study, eight candidate reference genes were analyzed in A. simplex s. s. at two different developmental stages: L3 and L4. The expression stability of these genes was assessed by geNorm and NormFinder softwares. Results In general, our results identified translation elongation factor 1α (ef-1α) and peptidyl-prolyl isomerase 12 (ppi12) as the most stable genes in L3 and L4 developmental stages of A. simplex s. s. Validation of the selected reference genes was performed by profiling the expression of the nuclear hormone receptor gene (nhr 48) in different developmental stages. Conclusions This first analysis selecting suitable reference genes for RT-qPCR in A. simplex s. s. will facilitate future functional analyses and deep mining of genetic resources in this parasitic nematode.

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Validation of Real-time PCR Reference Genes of Muscle Metabolism in Harvested Spiny-Cheek Crayfish (Faxonius limosus) Exposed to Seasonal Variation

Animals ◽

10.3390/ani10071140 ◽

2020 ◽

Vol 10 (7) ◽

pp. 1140

Author(s):

Natalia Śmietana ◽

Remigiusz Panicz ◽

Małgorzata Sobczak ◽

Piotr Eljasik ◽

Przemysław Śmietana

Keyword(s):

Real Time ◽

Calcium Binding ◽

Reference Genes ◽

Target Genes ◽

Arginine Kinase ◽

Elongation Factor ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Systematic Analysis ◽

Key Species

Real-time quantitative reverse transcription PCR (RT-qPCR) is a sensitive and broadly used technique of assessing gene activity. To obtain a reliable result, stably expressed reference genes are essential for normalization of transcripts in various samples. To our knowledge, this is the first systematic analysis of reference genes for normalization of RT-qPCR data in spiny-cheek crayfish (Faxonius limosus). In this study, expression of five candidate reference genes (actb, β-actin; gapdh, glyceraldehyde-3-phosphate dehydrogenase; eif, eukaryotic translation initiation factor 5a; ef-1α, elongation factor-1α; and tub, α-tubulin) in muscle samples from male and female F. limosus in spring and autumn was analyzed. Additionally, the most stable reference genes were used for accurate normalization of five target genes, i.e., tnnc, troponin c; ak, arginine kinase; fr, ferritin; ccbp-23, crustacean calcium-binding protein 23; and actinsk8, skeletal muscle actin 8. Results obtained using the geNorm and NormFinder algorithms showed high consistency, and differences in the activity of the selected actb with eif genes were successfully identified. The spring and autumn activities of the target genes (except ak) in the muscle tissue of males and females differed significantly, showing that both sexes are immensely involved in an array of breeding behaviors in spring, and females intensively recover in the autumn season. Characterization of first reference genes in spiny-cheek crayfish will facilitate more accurate and reliable expression studies in this key species.

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Identification of miR-328-3p as an endogenous reference gene for the normalization of miRNA expression data from patients with Diabetic Retinopathy

Scientific Reports ◽

10.1038/s41598-019-56172-w ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Marcelle SanJuan Ganem Prado ◽

Thaline Cunha de Goes ◽

Mirthz Lemos de Jesus ◽

Lucilla Silva Oliveira Mendonça ◽

Jadson Santos Nascimento ◽

...

Keyword(s):

Diabetic Retinopathy ◽

Mirna Expression ◽

Reference Genes ◽

Data Interpretation ◽

Stable Gene ◽

Internal Reference ◽

Expression Levels ◽

Rna Molecules ◽

Gene Normalization ◽

Mirna Expression Data

AbstractDiabetic Retinopathy, the main cause of visual loss and blindness among working population, is a complication of Diabetes mellitus (DM), which has been described as a major public health challenge, so it is important to identify biomarkers to predict and to stratify patient´s possibility for developing DR. MicroRNAs (miRNAs) are small non-coding RNA molecules that have showed to be promising disease biomarkers and association of miRNAs with the possibility to develop DR has been reported. However, evaluating miRNA expression involves normalization of RT-qPCR data using internal reference genes that should be properly determined, considering their impact on expression levels calculation and, until date, there is no unanimity on reference miRNAs for the investigation of circulating miRNAs in DR. We aimed to estimate the appropriateness of a group of miRNAs as normalizers to identify which might be considered steady internal reference genes in expression studies on DR plasma samples. Expression levels of candidates were analyzed in 60 healthy controls, 48 DM without DR patients and 62 DR patients with two statistical tools: NormFinder and RefFinder. MiR-328-3p was the most stable gene and we also investigated the effect of gene normalization, demonstrating that different normalization strategies have important implications for accurate data interpretation.

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Comparison of Reliable Reference Genes Following Different Hormone Treatments by Various Algorithms for qRT-PCR Analysis of Metasequoia

International Journal of Molecular Sciences ◽

10.3390/ijms20010034 ◽

2018 ◽

Vol 20 (1) ◽

pp. 34 ◽

Cited By ~ 3

Author(s):

Jing-Jing Wang ◽

Shuo Han ◽

Weilun Yin ◽

Xinli Xia ◽

Chao Liu

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Elongation Factor ◽

Pcr Analysis ◽

Qrt Pcr ◽

Gene Normalization ◽

Accurate Normalization ◽

Suitable Reference ◽

Different Tissues ◽

Gene Expression Levels

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the most sensitive technique for evaluating gene expression levels. Choosing appropriate reference genes for normalizing target gene expression is important for verifying expression changes. Metasequoia is a high-quality and economically important wood species. However, few systematic studies have examined reference genes in Metasequoia. Here, the expression stability of 14 candidate reference genes in different tissues and following different hormone treatments were analyzed using six algorithms. Candidate reference genes were used to normalize the expression pattern of FLOWERING LOCUS T and pyrabactin resistance-like 8. Analysis using the GrayNorm algorithm showed that ACT2 (Actin 2), HIS (histone superfamily protein H3) and TATA (TATA binding protein) were stably expressed in different tissues. ACT2, EF1α (elongation factor-1 alpha) and HIS were optimal for leaves treated with the flowering induction hormone solution, while Cpn60β (60-kDa chaperonin β-subunit), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and HIS were the best reference genes for treated buds. EF1α, HIS and TATA were useful reference genes for accurate normalization in abscisic acid-response signaling. Our results emphasize the importance of validating reference genes for qRT-PCR analysis in Metasequoia. To avoid errors, suitable reference genes should be used for different tissues and hormone treatments to increase normalization accuracy. Our study provides a foundation for reference gene normalization when analyzing gene expression in Metasequoia.

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Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization inIris. lacteavar.chinensisRoots under Cadmium, Lead, and Salt Stress Conditions

The Scientific World JOURNAL ◽

10.1155/2014/532713 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 7

Author(s):

Chun-Sun Gu ◽

Liang-qin Liu ◽

Chen Xu ◽

Yan-hai Zhao ◽

Xu-dong Zhu ◽

...

Keyword(s):

Gene Expression ◽

Salt Stress ◽

Real Time ◽

Reference Genes ◽

Elongation Factor ◽

Stress Conditions ◽

Translation Initiation Factor ◽

Translation Elongation Factor ◽

Translation Elongation ◽

Wide Range

Quantitative real time PCR (RT-qPCR) has emerged as an accurate and sensitive method to measure the gene expression. However, obtaining reliable result depends on the selection of reference genes which normalize differences among samples. In this study, we assessed the expression stability of seven reference genes, namely, ubiquitin-protein ligase UBC9 (UBC), tubulin alpha-5 (TUBLIN), eukaryotic translation initiation factor (EIF-5A), translation elongation factor EF1A (EF1α), translation elongation factor EF1B (EF1b), actin11 (ACTIN), and histone H3 (HIS), inIris. lacteavar.chinensis(I. lacteavar.chinensis) root when the plants were subjected to cadmium (Cd), lead (Pb), and salt stress conditions. All seven reference genes showed a relatively wide range of threshold cycles (Ct) values in different samples. GeNorm and NormFinder algorithms were used to assess the suitable reference genes. The results from the two software units showed thatEIF-5AandUBCwere the most stable reference genes across all of the tested samples, whileTUBLINwas unsuitable as internal controls.I. lacteavar.chinensisis tolerant to Cd, Pb, and salt. Our results will benefit future research on gene expression in response to the three abiotic stresses.

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