Identification of stable reference gene for qPCR studies in common wheat (Triticum aestivum L.) seedlings under short-term osmotic stress

Abstract BackgroundQuantitative PCR (qPCR) is one of the most common and accurate methods of gene expression analysis. However, the biggest challenge for this kind of examinations is normalization of the results, which requires the application of dependable internal controls. The selection of appropriate reference genes (RGs) is one of the most crucial points in qPCR data analysis and for correct assessment of gene expression. Because of the fact that many reports indicate that the expression profiles of typically used RGs can be unstable in certain experimental conditions, species or tissues, reference genes with stable expression levels should be selected individually for each experiment. In this study, we analysed a set of ten candidate RGs for wheat seedlings under short-term drought stress. Our tests included five ‘traditional’ RGs (GAPDH, ACT, UBI, TUB, and TEF1) and five novel genes developed by the RefGenes tool from the Genevestigator database.ResultsExpression stability was assessed using five different algorithms: geNorm, NormFinder, BestKeeper, RefFinder and the delta Ct method. In the final ranking, we identified three genes: CJ705892, ACT, and UBI, as the best candidates for housekeeping genes. However, our data indicated a slight variation between the different algorithms that were used. We revealed that the novel gene CJ705892, obtained by means of in silico analysis, showed the most stable expression in the experimental tissue and condition.ConclusionsOur results support the statement, that novel genes selected for certain experimental conditions have a more stable level of expression in comparison to routinely applied RGs, like genes encoding actin, tubulin or GAPDH. Selected CJ705892 gene can be used as a housekeeping gene in the expression analysis in wheat seedlings under short-term drought. The results of our study will be useful for subsequent analyses of gene expression in wheat tissues subjected to drought.

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Identification of stable reference genes for qPCR studies in common wheat (Triticum aestivum L.) seedlings under short-term drought stress

10.21203/rs.2.18396/v3 ◽

2020 ◽

Author(s):

Karolina Dudziak ◽

Magdalena Sozoniuk ◽

Hubert Szczerba ◽

Adam Kuzdraliński ◽

Krzysztof Kowalczyk ◽

...

Keyword(s):

Gene Expression ◽

Drought Stress ◽

Expression Analysis ◽

Reference Genes ◽

Stable Expression ◽

Slight Variation ◽

Short Term ◽

Novel Genes ◽

Experimental Conditions ◽

Wheat Seedlings

Abstract Background Quantitative PCR (qPCR) is one of the most common and accurate methods of gene expression analysis. However, the biggest challenge for this kind of examinations is normalization of the results, which requires the application of dependable internal controls. The selection of appropriate reference genes (RGs) is one of the most crucial points in qPCR data analysis and for correct assessment of gene expression. Because of the fact that many reports indicate that the expression profiles of typically used RGs can be unstable in certain experimental conditions, species or tissues, reference genes with stable expression levels should be selected individually for each experiment. In this study, we analysed a set of ten candidate RGs for wheat seedlings under short-term drought stress. Our tests included five ‘traditional’ RGs (GAPDH, ACT, UBI, TUB, and TEF1) and five novel genes developed by the RefGenes tool from the Genevestigator database.Results Expression stability was assessed using five different algorithms: geNorm, NormFinder, BestKeeper, RefFinder and the delta Ct method. In the final ranking, we identified three genes: CJ705892, ACT, and UBI, as the best candidates for housekeeping genes. However, our data indicated a slight variation between the different algorithms that were used. We revealed that the novel gene CJ705892, obtained by means of in silico analysis, showed the most stable expression in the experimental tissue and condition. Conclusions Our results support the statement, that novel genes selected for certain experimental conditions have a more stable level of expression in comparison to routinely applied RGs, like genes encoding actin, tubulin or GAPDH. Selected CJ705892 gene can be used as a housekeeping gene in the expression analysis in wheat seedlings under short-term drought. The results of our study will be useful for subsequent analyses of gene expression in wheat tissues subjected to drought.

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Identification of stable reference genes for qPCR studies in common wheat (Triticum aestivum L.) seedlings under short-term drought stress

10.21203/rs.2.18396/v1 ◽

2019 ◽

Author(s):

Karolina Dudziak ◽

Magdalena Sozoniuk ◽

Hubert Szczerba ◽

Adam Kuzdraliński ◽

Krzysztof Kowalczyk ◽

...

Keyword(s):

Gene Expression ◽

Drought Stress ◽

Expression Analysis ◽

Reference Genes ◽

Stable Expression ◽

Slight Variation ◽

Short Term ◽

Novel Genes ◽

Experimental Conditions ◽

Wheat Seedlings

Abstract Background: Quantitative PCR (qPCR) is one of the most common and accurate methods of gene expression analysis. However, the biggest challenge for this kind of examinations is normalization of the results, which requires the application of dependable internal controls. The selection of appropriate reference genes (RGs) is one of the most crucial points in qPCR data analysis and for correct assessment of gene expression. Because of the fact that many reports indicate that the expression profiles of typically used RGs can be unstable in certain experimental conditions, species or tissues, reference genes with stable expression levels should be selected individually for each experiment. In this study, we analysed a set of ten candidate RGs for wheat seedlings under short-term drought stress. Our tests included five ‘traditional’ RGs (GAPDH, ACT, UBI, TUB, and TEF1) and five novel genes developed by the RefGenes tool from the Genevestigator database.Results: Expression stability was assessed using five different algorithms: geNorm, NormFinder, BestKeeper, RefFinder and the delta Ct method. In the final ranking, we identified three genes: CJ705892, ACT, and UBI, as the best candidates for housekeeping genes. However, our data indicated a slight variation between the different algorithms that were used. We revealed that the novel gene CJ705892, obtained by means of in silico analysis, showed the most stable expression in the experimental tissue and condition. Conclusions: Our results support the statement, that novel genes selected for certain experimental conditions have a more stable level of expression in comparison to routinely applied RGs, like genes encoding actin, tubulin or GAPDH. Selected CJ705892 gene can be used as a housekeeping gene in the expression analysis in wheat seedlings under short-term drought. The results of our study will be useful for subsequent analyses of gene expression in wheat tissues subjected to drought.

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Identification of stable reference genes for qPCR studies in common wheat (Triticum aestivum L.) seedlings under short-term drought stress

10.21203/rs.2.18396/v2 ◽

2020 ◽

Author(s):

Karolina Dudziak ◽

Magdalena Sozoniuk ◽

Hubert Szczerba ◽

Adam Kuzdraliński ◽

Krzysztof Kowalczyk ◽

...

Keyword(s):

Gene Expression ◽

Drought Stress ◽

Expression Analysis ◽

Reference Genes ◽

Stable Expression ◽

Slight Variation ◽

Short Term ◽

Novel Genes ◽

Experimental Conditions ◽

Wheat Seedlings

Abstract Background Quantitative PCR (qPCR) is one of the most common and accurate methods of gene expression analysis. However, the biggest challenge for this kind of examinations is normalization of the results, which requires the application of dependable internal controls. The selection of appropriate reference genes (RGs) is one of the most crucial points in qPCR data analysis and for correct assessment of gene expression. Because of the fact that many reports indicate that the expression profiles of typically used RGs can be unstable in certain experimental conditions, species or tissues, reference genes with stable expression levels should be selected individually for each experiment. In this study, we analysed a set of ten candidate RGs for wheat seedlings under short-term drought stress. Our tests included five ‘traditional’ RGs (GAPDH, ACT, UBI, TUB, and TEF1) and five novel genes developed by the RefGenes tool from the Genevestigator database. Results Expression stability was assessed using five different algorithms: geNorm, NormFinder, BestKeeper, RefFinder and the delta Ct method. In the final ranking, we identified three genes: CJ705892, ACT, and UBI, as the best candidates for housekeeping genes. However, our data indicated a slight variation between the different algorithms that were used. We revealed that the novel gene CJ705892, obtained by means of in silico analysis, showed the most stable expression in the experimental tissue and condition. Conclusions Our results support the statement, that novel genes selected for certain experimental conditions have a more stable level of expression in comparison to routinely applied RGs, like genes encoding actin, tubulin or GAPDH. Selected CJ705892 gene can be used as a housekeeping gene in the expression analysis in wheat seedlings under short-term drought. The results of our study will be useful for subsequent analyses of gene expression in wheat tissues subjected to drought.

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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 normalization of qRT-PCR data to study secondary metabolite related genes in industrial hemp

10.21203/rs.3.rs-394417/v1 ◽

2021 ◽

Author(s):

Michihito Deguchi ◽

Shobha Potlakayala ◽

Zachary Spuhler ◽

Hannah George ◽

Vijay Sheri ◽

...

Keyword(s):

Gene Expression ◽

Heavy Metal ◽

Expression Analysis ◽

Reference Genes ◽

Cannabis Sativa ◽

Expression Profiles ◽

Expression Patterns ◽

Qrt Pcr ◽

Differential Gene ◽

Industrial Hemp

Abstract Industrial hemp (Cannabis sativa L.) is a dioecious crop widely known for its production of phytocannabinoids, flavonoids, and terpenes. In the past two years since its legalization, there has been significant interest in researching this important crop for pharmaceutical applications. Although many scientific reports have demonstrated gene expression analysis of hemp through OMICs approaches, accurate validation of omics data cannot be performed because of lack of reliable reference genes for normalization of qRT-PCR data. The differential gene expression patterns of 13 candidate reference genes under osmotic, heavy metal, hormonal, and UV stress were evaluated through four software packages: geNorm, NormFinder, BestKeeper, and RefFinder. The EF-1a ranked as the most stable reference gene across all stresses, TUB was the most stable under osmotic stress, and TATA was the most stable under both heavy metal and hormonal stress. The expression profiles of two cannabinoid pathway genes, AAE1 and THCAS, using the two most stable and single least stable reference genes confirmed that two most stables genes were apt for normalization of gene expression data. This work will contribute to the future studies on the expression analysis of hemp genes regulating the synthesis, transport and accumulation of secondary metabolites.

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Genome Based Search for Reference Genes for Gene Expression Analysis in Oral Cancer: a Data Science Driven Approach

10.21203/rs.2.10145/v1 ◽

2019 ◽

Author(s):

Nehanjali Dwivedi ◽

Sujan K Dhar ◽

G Charitha ◽

Moni Abraham Kuriakose ◽

Amritha Suresh ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Expression Analysis ◽

Reference Genes ◽

Data Science ◽

Gene Expression Analysis ◽

The Cancer Genome Atlas ◽

Experimental Conditions ◽

Statistical Measures ◽

Different Types

Abstract Background Quantitative real time PCR (qPCR) remains by far the most cost-effective, fast yet sensitive technique to check the gene expression levels in various systems. The traditionally used reference genes over the years were found to be regulated heavily based on sample sources and/or experimental conditions. This paper therefore presents a data science driven -omic approach for selection of reference genes from ~60,000 candidates from The Cancer Genome Atlas (TCGA) and Broad Institute Cancer Cell Line Encyclopaedia (CCLE) for gene expression studies in head and neck squamous cell carcinoma (HNSCC). mRNA-sequencing data of 500 patient samples and 33 cell lines from publicly available databases were analysed to assess stability of genes in terms of multiple statistical measures. A final set of 12 candidate genes were studied in the Indian set of data in Gene Expression Omnibus (GEO) and validated experimentally using qPCR in 35 different types of samples from platforms like drug sensitive and resistant cell lines, normal and tumor samples, fibroblast and epithelial primary culture derived from HNSCC patients from India. Result The study lead to the choice of five most stable reference genes –TYW5, RIC8B, PLEKHA3, CEP57L1 and GPR89B across three experimental platforms. Conclusion In addition to providing a set of five most stable reference genes for future gene expression analysis experiments across different types of samples in HNSCC, the study also presents an objective framework for assessing reference genes for other disease areas as well.

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Reference Gene Selection for qPCR Normalization ofKosteletzkya virginicaunder Salt Stress

BioMed Research International ◽

10.1155/2015/823806 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 8

Author(s):

Xiaoli Tang ◽

Hongyan Wang ◽

Chuyang Shao ◽

Hongbo Shao

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Gene Selection ◽

Expression Profiles ◽

Elongation Factor ◽

Gene Expression Profiles ◽

Stable Reference Gene ◽

Experimental Conditions ◽

Suitable Reference

Kosteletzkya virginica(L.) is a newly introduced perennial halophytic plant. Presently, reverse transcription quantitative real-time PCR (qPCR) is regarded as the best choice for analyzing gene expression and its accuracy mainly depends on the reference genes which are used for gene expression normalization. In this study, we employed qPCR to select the most stable reference gene inK. virginicawhich showed stable expression profiles under our experimental conditions. The candidate reference genes were 18S ribosomal RNA (18SrRNA),β-actin (ACT),α-tubulin (TUA), and elongation factor (EF). We tracked the gene expression profiles of the candidate genes and analyzed their stabilities through BestKeeper, geNorm, and NormFinder software programs. The results of the three programs were identical and18SrRNAwas assessed to be the most stable reference gene in this study. However,TUAwas identified to be the most unstable. Our study proved again that the traditional reference genes indeed displayed a certain degree of variations under given experimental conditions. Importantly, our research also provides guidance for selecting most suitable reference genes and lays the foundation for further studies inK. virginica.

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Validation of Novel Reference Genes for Reverse Transcription Quantitative Real-Time PCR in Drought-Stressed Sugarcane

The Scientific World JOURNAL ◽

10.1155/2014/357052 ◽

2014 ◽

Vol 2014 ◽

pp. 1-12 ◽

Cited By ~ 9

Author(s):

Roberta Lane de Oliveira Silva ◽

Manassés Daniel Silva ◽

José Ribamar Costa Ferreira Neto ◽

Claudia Huerta de Nardi ◽

Sabrina Moutinho Chabregas ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Expression Profiles ◽

Acc Oxidase ◽

Quantitative Real Time Pcr ◽

Experimental Conditions ◽

Drought Tolerant ◽

Statistical Approaches ◽

Root Tissues ◽

Transcriptomic Studies

One of the most challenging aspects of RT-qPCR data analysis is the identification of reliable reference genes. Ideally, they should be neither induced nor repressed under different experimental conditions. To date, few reference genes have been adequately studied for sugarcane (Saccharumspp.) using statistical approaches. In this work, six candidate genes (αTUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) were tested for gene expression normalization of sugarcane root tissues from drought-tolerant and -sensitive accessions after continuous dehydration (24 h). By undergoing different approaches (GeNorm, NormFinder, and BestKeeper), it was shown that most of them could be used in combinations for normalization purposes, with the exception of SAMDC. Nevertheless three of them (H1,αTUB, and GAPDH) were considered the most reliable reference genes. Their suitability as reference genes validated the expression profiles of two targets (AS and PFPα1), related to SuperSAGE unitags, in agreement with results revealed by previousin silicoanalysis. The other two sugarcane unitags (ACC oxidase and PIP1-1), after salt stress (100 mM NaCl), presented their expressions validated in the same way. In conclusion, these reference genes will be useful for dissecting gene expression in sugarcane roots under abiotic stress, especially in transcriptomic studies using SuperSAGE or RNAseq approaches.

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Selection and Validation of Reference Genes for Quantitative Real-Time PCR Expression Analysis of Candidate Genes in Carposina sasakii (Lepidoptera: Carposinidae)

Biotechnology Journal International ◽

10.9734/bji/2020/v24i630124 ◽

2020 ◽

pp. 75-85

Author(s):

Zuobing Yan ◽

Yongli Li ◽

Zhou Zhou ◽

Yongan Zhang ◽

Liangjian Qu

Keyword(s):

Gene Expression ◽

Real Time ◽

Expression Analysis ◽

Real Time Pcr ◽

Reference Genes ◽

Developmental Stages ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Quantitative Real Time Pcr ◽

Carposina Sasakii

Carposina sasakii is one of the most important pests on the quality of stone and pome fruits. Investigation of a gene expression level in the species is hampered because of the gap of validated reference genes. The expression variation in the transcription levels of eight candidate reference genes, Actin (ACT), Tubulinbeta-1 (TUB), Ribosomal protein 49 (RP49), Elongation factor1-alpha (EF-1a), Elongation factor1-b (EF-1b), Elongation factor1-d (EF-1d), Ribosomal proteinL13 (RPL13) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were analyzed by quantitative real-time PCR (qPCR). The stability and ranking of these gene expression profiles in three organ types (head, thorax and abdomen), three developmental stages (larva, pupa and moth), and five diapause states (non-diapause, pre-diapause, diapause 0 d, diapause 20 d and diapause 60 d) were assessed using two algorithm-based methods, geNorm and NormFinder. EF-1a, ACT and GAPDH were evaluated to be the three stable reference genes based on the important observations and comprehensive analysis, whereas TUB and EF-1b showed low expression stability. Best gene combinations for different qPCR analysis in C. sasakii could be chosen from the three stable reference genes, the using of two reference genes is sufficient to effectively normalize qPCR data in C. sasakii. The study laid the foundation for gene expression analysis in C. sasakii and provided new information for the selection of reference genes.

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Evaluation of candidate reference genes for gene expression analysis in the brassica leaf beetle, Phaedon brassicae (Coleoptera: Chrysomelidae)

PLoS ONE ◽

10.1371/journal.pone.0251920 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0251920

Author(s):

Long Ma ◽

Ting Jiang ◽

Xiangya Liu ◽

Haijun Xiao ◽

Yingchuan Peng ◽

...

Keyword(s):

Gene Expression ◽

Thermal Stress ◽

Expression Analysis ◽

Reference Genes ◽

Gene Expression Analysis ◽

Developmental Stages ◽

Sequence Data ◽

Leaf Beetle ◽

Experimental Conditions ◽

Molecular Studies

The brassica leaf beetle Phaedon brassicae is a notorious defoliator of cruciferous vegetables. However, few molecular studies of this pest have been conducted due to limited sequence data. Recently, RNA sequencing has offered a powerful platform to generate numerous transcriptomic data, which require RT-qPCR to validate target gene expression. The selection of reliable reference genes to normalize RT-qPCR data is a prerequisite for gene expression analysis. In the present study, the expression stabilities of eight candidate reference genes under biotic conditions (development stages and various tissues) and abiotic perturbations (thermal stress and pesticide exposure) were evaluated using four different statistical algorithms. The optimal suites of reference genes were recommended for the respective experimental conditions. For tissue expression analysis, RPL32 and EF-1α were recommended as the suitable reference genes. RPL19 and TBP were the optimal reference genes across different developmental stages. RPL32 and TBP were identified as the most suitable references for thermal stress. Furthermore, RPL32 and RPL19 were ranked as the best references for insecticide exposure. This work provides a systematic exploration of the optimal reference genes for the respective experimental conditions, and our findings would facilitate molecular studies of P. brassicae.

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