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 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 quantitative gene expression normalization in Taxus spp.

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.

scholarly journals Expression Analysis of XTH in Stem Swelling of Stem Mustard and Selection of Reference Genes

Genes ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 113 ◽  
Author(s):  
Mengyao Li ◽  
Fangjie Xie ◽  
Qi He ◽  
Jie Li ◽  
Jiali Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Expression Patterns ◽  
Regulatory Mechanisms ◽  
Future Research ◽  
Transcriptome Data ◽  
Accurate Analysis ◽  
Report Analysis ◽  
The Stability ◽  
Selection Of

Accurate analysis of gene expression requires selection of appropriate reference genes. In this study, we report analysis of eight candidate reference genes (ACTIN, UBQ, EF-1α, UBC, IF-4α, TUB, PP2A, and HIS), which were screened from the genome and transcriptome data in Brassica juncea. Four statistical analysis softwares geNorm, NormFinder, BestKeeper, and RefFinder were used to test the reliability and stability of gene expression of the reference genes. To further validate the stability of reference genes, the expression levels of two CYCD3 genes (BjuB045330 and BjuA003219) were studied. In addition, all genes in the xyloglucan endotransglucosylase/hydrolase (XTH) family were identified in B. juncea and their patterns at different periods of stem enlargement were analyzed. Results indicated that UBC and TUB genes showed stable levels of expression and are recommended for future research. In addition, XTH genes were involved in regulation of stem enlargement expression. These results provide new insights for future research aiming at exploring important functional genes, their expression patterns and regulatory mechanisms for mustard development.

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

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.

scholarly journals Selection and validation reference genes for qRT-PCR normalization in different cultivars during fruit ripening and softening of peach (Prunus persica)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shuanghong You ◽  
Ke Cao ◽  
Changwen Chen ◽  
Yong Li ◽  
Jinlong Wu ◽  
...  
Keyword(s):  
Reference Gene ◽  
Fruit Ripening ◽  
Reference Genes ◽  
Prunus Persica ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Regulatory Mechanisms ◽  
Suitable Reference Gene ◽  
Future Research ◽  
Qrt Pcr

AbstractQuantitative real-time PCR (qRT-PCR) has been emerged as an effective method to explore the gene function and regulatory mechanisms. However, selecting appropriate reference gene (s) is a prerequisite for obtaining accurate qRT-PCR results. Peach is one of important fruit in Rosaceae and is widely cultivated worldwide. In this study, to explore reliable reference gene (s) in peach with different types during fruit ripening and softening (S1–S4), nine candidate reference genes (EF-1α, GAPDH, TBP, UBC, eIF-4α, TUB-A, TUB-B, ACTIN, and HIS) were selected from the whole-genome data. Then, the expression levels of the nine selected genes were detected using qRT-PCR in three peach types, including ‘Hakuho’ (melting type), ‘Xiacui’ (stony hard type), ‘Fantasia’ and ‘NJC108’ (non-melting type) cultivars were detected using qRT-PCR. Four software (geNorm, NormFinder, BestKeeper and RefFinder) were applied to evaluate the expression stability of these candidate reference genes. Gene expression was characterized in different peach types during fruit ripening and softening stages. The overall performance of each candidate in all samples was evaluated. The Actin gene (ACTIN) was a suitable reference gene and displayed excellent stability in ‘Total’ set, ‘Hakuho’ samples, S3 and S4 fruit developmental stages. Ubiquitin C gene (UBC) showed the best stability in most independent samples, including ‘Fantasia’, ‘NJC108’, S2 sets. Elongation factor-1α gene (EF-1α) was the most unstable gene across the set of all samples, ‘NJC108’ and S2 sets, while showed the highest stability in ‘Xiacui’ samples. The stability of candidate reference genes was further verified by analyzing the relative expression level of ethylene synthase gene of Prunus persica (PpACS1) in fruit ripening and softening periods of ‘Hakuho’. Taken together, the results from this study provide a basis for future research on the mining of important functional genes, expression patterns and regulatory mechanisms in peach.

scholarly journals Selection and Validation of Reference Genes for qRT-PCR Analysis in the Oil-Rich Tuber Crop Tiger Nut (Cyperus esculentus) Based on Transcriptome Data

2021 ◽  
Vol 22 (5) ◽  
pp. 2569
Author(s):  
Xue Bai ◽  
Tao Chen ◽  
Yuan Wu ◽  
Mingyong Tang ◽  
Zeng-Fu Xu
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Cyperus Esculentus ◽  
Transcriptome Data ◽  
Qrt Pcr ◽  
Below Ground ◽  
The Stability

Tiger nut (Cyperus esculentus), a perennial C4 plant of the Cyperaceae family, is an unconventional crop that is distinguished by its oil-rich tubers, which also possesses the advantages of strong resistance, wide adaptability, short life periods, and large biomass. To facilitate studies on gene expression in this species, we identified and validated a series of reference genes (RGs) based on transcriptome data, which can be employed as internal controls for qRT-PCR analysis in tiger nut. Fourteen putative candidate RGs were identified and evaluated across nine different tissues of two cultivars, and the RGs were analyzed using three different algorithms (geNorm, NormFinder, and BestKeeper). The stability rankings of the candidate RGs were merged into consensus lists with RankAggreg. For the below-ground storage organ of tiger nut, the optimal RGs were TUB4 and UCE2 in different developmental stages of tubers. UCE2 and UBL5 were the most stably expressed RGs among all tissues, while Rubisco and PGK exhibited the lowest expression stability. UCE2, UBL5 and Rubisco were compared to normalize the expression levels of the caleosin (CLO) and diacylglycerol acyltransferase 2-2 (DGAT2-2) genes across the same tissues. Our results showed that the RGs identified in this study, which exhibit more uniform expression patterns, may be utilized for the normalization of qRT-PCR results, promoting further research on gene expression in various tissues of tiger nut.

scholarly journals Selection and Evaluation of Reference Genes for Quantitative Real-Time Reverse-Transcriptase PCR under Cold Stress in Eggplant (Solanum melongena L.) during Fruit Development

2020 ◽  
Author(s):  
Jinkun Yang ◽  
Ying Zhang ◽  
Yong Lian ◽  
Yuhui Chen ◽  
Fuzhong Liu
Keyword(s):  
Gene Expression ◽  
Cold Stress ◽  
Cold Tolerance ◽  
Fruit Development ◽  
Reference Genes ◽  
Sequence Data ◽  
Expression Patterns ◽  
Solanum Melongena ◽  
Stable Gene ◽  
Qrt Pcr

Abstract Background Eggplant (Solanum melongena L.) is a thermophilic vegetable, and its yield and quality are often affected by cold stress. Therefore, identifying the key genes and mechanisms of cold tolerance has become a significant topic in eggplant. qRT-PCR has been widely used to analyse gene expression patterns, and reliable reference genes are necessary for this technique. Methods To select and evaluate suitable reference genes for qRT-PCR, 18 candidate genes selected from transcriptome sequence data were subjected to analysis of their expression stability under natural cold and normal temperature conditions. Four commonly used programs (geNorm, NormFinder, BestKeeper and RefFinder) were used to determine the stabilities of these genes. Results The results showed that D5, D4 and D1 were the three most stable reference genes among the 18 genes. Then, D5, D4 and D1 were compared with commonly used reference genes. The results showed that D5 was still the most stable gene, followed by APRT, D4, and Actin was the least stable gene. Conclusion D5, APRT and D4 were recommended as a reference gene combination for gene expression normalization under cold stress and at normal temperature during fruit development. Our results provide a molecular foundation for further research on the cold tolerance mechanism of eggplant.

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

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.

scholarly journals Validation of Reference Genes for Studying Different Abiotic Stresses in oat (Avena sativa L.) by RT-qPCR

Plants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1272
Author(s):  
Judit Tajti ◽  
Magda Pál ◽  
Tibor Janda
Keyword(s):  
Gene Expression ◽  
Avena Sativa ◽  
Abiotic Stresses ◽  
Reference Genes ◽  
Nutritional Value ◽  
Tissue Type ◽  
Future Research ◽  
Experimental Conditions ◽  
Expression Studies ◽  
Gene Expression Studies

Oat (Avena sativa L.) is a widely cultivated cereal with high nutritional value and it is grown mainly in temperate regions. The number of studies dealing with gene expression changes in oat continues to increase, and to obtain reliable RT-qPCR results it is essential to establish and use reference genes with the least possible influence caused by experimental conditions. However, no detailed study has been conducted on reference genes in different tissues of oat under diverse abiotic stress conditions. In our work, nine candidate reference genes (ACT, TUB, CYP, GAPD, UBC, EF1, TBP, ADPR, PGD) were chosen and analysed by four statistical methods (GeNorm, Normfinder, BestKeeper, RefFinder). Samples were taken from two tissues (leaves and roots) of 13-day-old oat plants exposed to five abiotic stresses (drought, salt, heavy metal, low and high temperatures). ADPR was the top-rated reference gene for all samples, while different genes proved to be the most stable depending on tissue type and treatment combinations. TUB and EF1 were most affected by the treatments in general. Validation of reference genes was carried out by PAL expression analysis, which further confirmed their reliability. These results can contribute to reliable gene expression studies for future research in cultivated oat.

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