scholarly journals Selection and validation of reference genes for normalization of qRT-PCR data to study secondary metabolite related genes in industrial hemp

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.

scholarly journals Selection and validation of reference genes for normalization of qRT-PCR data to study the cannabinoid pathway genes in industrial hemp

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260660
Author(s):  
Michihito Deguchi ◽  
Shobha Potlakayala ◽  
Zachary Spuhler ◽  
Hannah George ◽  
Vijay Sheri ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heavy Metal ◽  
Reference Genes ◽  
Expression Patterns ◽  
Heavy Metal Stress ◽  
Future Research ◽  
Qrt Pcr ◽  
Differential Gene ◽  
Expression Characterization ◽  
Industrial Hemp

There has been significant interest in researching the pharmaceutical applications of Industrial hemp since its legalization three years ago. The crop is mostly dioecious and known for its production of phytocannabinoids, flavonoids, and terpenes. Although many scientific reports have showed gene expression analysis of hemp through OMICs approaches, unreliable reference genes for normalization of qRT-PCR data make it difficult to validate the OMICs data. Four software packages: geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the differential gene expression patterns of 13 candidate reference genes under osmotic, heavy metal, hormonal, and UV stresses. EF-1α 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 stress and hormonal stimuli. The expression patterns of two cannabinoid pathway genes, AAE1 and CBDAS, were used to validate the reliability of the selected reference genes. This work provides useful information for gene expression characterization in hemp and future research in the synthesis, transport, and accumulation of secondary metabolites.

scholarly journals Identification and validation of miRNA reference genes in poplar under pathogen stress

2021 ◽  
Author(s):  
Lichun Zhang ◽  
Xiaoqian Yang ◽  
Yiyi Yin ◽  
Jinxing Wang ◽  
Yanwei Wang
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Small Rna ◽  
Reference Genes ◽  
Expression Profiles ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Internal Standards ◽  
Qrt Pcr ◽  
Canker Pathogen

Abstract Quantitative real time polymerase chain reaction (qRT-PCR) is a common method to analyze gene expression. Due to differences in RNA quantity, quality, and reverse transcription efficiency between qRT-PCR samples, reference genes are used as internal standards to normalize gene expression. However, few universal genes especially miRNAs have been identified as reference so far. Therefore, it is essential to identify reference genes that can be used across various experimental conditions, stress treatments, or tissues. In this study, 14 microRNAs (miRNAs) and 5.8S rRNA were assessed for expression stability in poplar trees infected with canker pathogen. Using three reference gene analysis programs, we found that miR156g and miR156a exhibited stable expression throughout the infection process. miR156g and miR156a were then tested as internal standards to measure the expression of miR1447 and miR171c, and the results were compared to small RNA sequencing (RNA-seq) data. We found that when miR156a was used as the reference gene, the expression of miR1447 and miR171c were consistent with the small RNA-seq expression profiles. Therefore, miR156a was the most stable miRNAs examined in this study, and could be used as a reference gene in poplar under canker pathogen stress, which should enable comprehensive comparisons of miRNAs expression and avoid the bias caused by different lenth between detected miRNAs and traditional referece genes. The present study has expanded the miRNA reference genes available for gene expression studies in trees under biotic stress.

scholarly journals RankerGUI: A Computational Framework to Compare Differential Gene Expression Profiles Using Rank Based Statistics

2019 ◽  
Vol 20 (23) ◽  
pp. 6098 ◽  
Author(s):  
Amarinder Singh Thind ◽  
Kumar Parijat Tripathi ◽  
Mario Rosario Guarracino
Keyword(s):  
Gene Expression ◽  
Differential Expression ◽  
Differential Gene Expression ◽  
Web Application ◽  
Cellular Response ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Gene Expression Profiles ◽  
Experimental Conditions ◽  
Differential Gene

The comparison of high throughput gene expression datasets obtained from different experimental conditions is a challenging task. It provides an opportunity to explore the cellular response to various biological events such as disease, environmental conditions, and drugs. There is a need for tools that allow the integration and analysis of such data. We developed the “RankerGUI pipeline”, a user-friendly web application for the biological community. It allows users to use various rank based statistical approaches for the comparison of full differential gene expression profiles between the same or different biological states obtained from different sources. The pipeline modules are an integration of various open-source packages, a few of which are modified for extended functionality. The main modules include rank rank hypergeometric overlap, enriched rank rank hypergeometric overlap and distance calculations. Additionally, preprocessing steps such as merging differential expression profiles of multiple independent studies can be added before running the main modules. Output plots show the strength, pattern, and trends among complete differential expression profiles. In this paper, we describe the various modules and functionalities of the developed pipeline. We also present a case study that demonstrates how the pipeline can be used for the comparison of differential expression profiles obtained from multiple platforms’ data of the Gene Expression Omnibus. Using these comparisons, we investigate gene expression patterns in kidney and lung cancers.

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

2020 ◽  
Author(s):  
Karolina Dudziak ◽  
Magdalena Sozoniuk ◽  
Andreas Börner ◽  
Hubert Szczerba ◽  
Adam Kuzdraliński ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Reference Genes ◽  
Expression Profiles ◽  
Stable Expression ◽  
Slight Variation ◽  
Short Term ◽  
Novel Genes ◽  
Experimental Conditions ◽  
Wheat Seedlings

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.

scholarly journals Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes

2020 ◽  
Author(s):  
Liz M. Florez ◽  
Reiny W. A. Scheper ◽  
Brent M. Fisher ◽  
Paul W. Sutherland ◽  
Matthew D. Templeton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Reference Genes ◽  
Time Course ◽  
Gene Expression Analysis ◽  
Virulence Genes ◽  
Functional Characterization ◽  
Post Inoculation ◽  
In Planta ◽  
Qrt Pcr

AbstractEuropean canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Real-time quantitative reverse transcription PCR (qRT-PCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate qRT-PCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.

scholarly journals Validation of Reference Genes for Quantitative Real-time PCR in Diaphanosoma Celebensis: For Chemical Exposure Conditions and Different Ages

2021 ◽  
Author(s):  
Young-Mi Lee ◽  
Soyeon In ◽  
Se-Joo Kim ◽  
Eun-Ji Won ◽  
Hayoung Cho ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Reference Genes ◽  
Expression Profiles ◽  
Chemical Exposure ◽  
Mrna Levels ◽  
Qrt Pcr ◽  
Different Ages ◽  
Diaphanosoma Celebensis

Abstract Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), a primary approach for evaluating gene expression, requires an appropriate normalization strategy to rule out variations in gene expression among samples. The best option is to use a reference gene whose expression level is stable across various experimental conditions to compare the mRNA levels of a target gene. However, there is limited information on how the reference gene is differentially expressed at different ages (growth) in small invertebrates with notable changes such as molting. In this study, expression profiles of nine candidate reference genes from the brackish water flea, Diaphanosoma celebensis, were evaluated under diverse exposure to toxicants and according to growth. As a result, four different algorithms showed similar stabilities of genes for chemical exposures in the case of limited conditions using the same developmental stage (e.g., adult), while the results according to age showed a significantly different pattern in suite of candidate reference genes. This affected the results of genes EcRA and GST, which are involved in development and detoxification mechanisms, respectively. Our finding is the first step towards establishing a standardized real-time qRT-PCR analysis of this environmentally important invertebrate that has potential for aquatic ecotoxicology, particularly in estuarine environments.

scholarly journals Selection and validation of reference genes desirable for gene expression analysis by qRT-PCR in MeJA-treated ginseng hairy roots

PLoS ONE ◽  
2019 ◽  
Vol 14 (12) ◽  
pp. e0226168 ◽  
Author(s):  
Li Li ◽  
Kangyu Wang ◽  
Mingzhu Zhao ◽  
Shaokun Li ◽  
Yue Jiang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Hairy Roots ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Qrt Pcr

Differential Gene Expression for Age Estimation of Forensically Important Sarcophaga peregrina (Diptera: Sarcophagidae) Intrapuparial

2019 ◽  
Vol 57 (1) ◽  
pp. 65-77 ◽  
Author(s):  
Yanjie Shang ◽  
Lipin Ren ◽  
Li Yang ◽  
Shiwen Wang ◽  
Wei Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Differential Gene Expression ◽  
Age Estimation ◽  
Reference Genes ◽  
Morphological Changes ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Accurate Determination ◽  
28S Rrna ◽  
Differential Gene

Abstract Sarcophaga peregrina is an important flesh fly species for estimating the minimum postmortem interval (PMImin) in forensic entomology. The accurate determination of the developmental age is a crucial task for using necrophagous sarcophagids to estimate PMImin. During larval development, the age determination is straight forward by the morphological changes and variation of length, weight, and width; however, the age estimation of sarcophagid intrapuparial is more difficult due to anatomical and morphological changes not being visible. The analysis of differentially expressed genes (DEGs) during sarcophagid metamorphosis is a potential method for age estimation of intrapuparial. In the present study, real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the differential gene expression level of S. peregrina intrapuparial in different constant temperatures (35°C, 25°C, and 15°C). In addition, the appropriate reference genes of S. peregrina were selected in the intrapuparial and at different temperatures to obtain reliable and valid gene expression profiles. The results indicated that two candidate genes (18S rRNA and 28S rRNA) were the most reliable reference genes, and four DEGs (Hsp90, A-alpha, AFP, AFBP) have the potential to be used to more accuracy estimate the age of S. peregrina intrapuparial.

scholarly journals Identification and Evaluation of Reference Genes for Normalization of Gene Expression in Developmental Stages, Sexes, and Tissues of Diaphania caesalis (Lepidoptera, Pyralidae)

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Zheng Wang ◽  
Qianqian Meng ◽  
Xi Zhu ◽  
Shiwei Sun ◽  
Aiqin Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Target Gene ◽  
Target Genes ◽  
Developmental Stages ◽  
Expression Profiles ◽  
Transcriptional Level ◽  
Internal Reference ◽  
Target Gene Expression ◽  
Qrt Pcr

Abstract Diaphania caesalis (Walker) is an important boring insect mainly distributed in subtropical and tropical areas and attacked tropical woody grain crops, such as starchy plants of Artocarpus. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful approach for investigating target genes expression profiles at the transcriptional level. However, the identification and selection of internal reference genes, which is often overlooked, is the most vital step before the analysis of target gene expression by qRT-PCR. So far, the reliable internal reference genes under a certain condition of D. caesalis have not been investigated. Therefore, this study evaluated the expression stability of eight candidate reference genes including ACT, β-TUB, GAPDH, G6PDH, RPS3a, RPL13a, EF1α, and EIF4A in different developmental stages, tissues and sexes using geNorm, NormFinder and BestKeeper algorithms. To verify the stability of the recommended internal reference genes, the expression levels of DcaeOBP5 were analyzed under different treatment conditions. The results indicated that ACT, RPL13a, β-TUB, RPS3a, and EF1α were identified as the most stable reference genes for further studies on target gene expression involving different developmental stages of D. caesalis. And ACT and EIF4A were recommended as stable reference genes for different tissues. Furthermore, ACT, EF1α, and RPS3a were ranked as the best reference genes in different sexes based on three algorithms. Our research represents the critical first step to normalize qRT-PCR data and ensure the accuracy of expression of target genes involved in phylogenetic and physiological mechanism at the transcriptional level in D. caesalia.

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