scholarly journals Selection and Validation of Reference Genes for the qRT-PCR Assays of Populus ussuriensis Gene Expression under Abiotic Stresses and Related ABA Treatment

Forests ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 476
Author(s):  
Ming Wei ◽  
Yingxi Chen ◽  
Mengqiu Zhang ◽  
Jingli Yang ◽  
Han Lu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cold Stress ◽  
Internal Control ◽  
Reference Genes ◽  
Target Genes ◽  
High Salinity ◽  
High Yield ◽  
Pcr Analysis ◽  
Qrt Pcr ◽  
Populus Ussuriensis

Populus ussuriensis Kom. is one of the most important tree species for forest renewal in the eastern mountainous areas of Northeast China due to its fast growth, high yield, and significant commercial and ecological value. The selection of optimal reference genes for the normalization of qRT-PCR data is essential for the analysis of relative gene expression. In this study, fourteen genes were selected and assessed for their expression stability during abiotic stress (drought, high salinity, and cold stress) and after the treatment with the drought-related hormone ABA. Three algorithms were used, geNorm, NormFinder, and BestKeeper, and a comprehensive ranking of candidate reference genes was produced based on their output. The most appropriate reference genes were UBQ10 and RPL24 for drought and ABA treatment, UBQ10 and TUB3 for cold stress, and UBQ10 and 60S rRNA for high salinity. Overall, UBQ10 was the most stable reference gene for use as an internal control, whereas PP2A was the least stable. The expression of two target genes (P5CS2 and GI) was used to further verify that the selected reference genes were suitable for gene expression normalization. This work comprehensively assesses the stability of reference genes in Populus ussuriensis and identifies suitable reference genes for normalization during qRT-PCR analysis.

scholarly journals Reference gene selection for qRT-PCR assays inStellera chamaejasmesubjected to abiotic stresses and hormone treatments based on transcriptome datasets

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4535 ◽  
Author(s):  
Xin Liu ◽  
Huirui Guan ◽  
Min Song ◽  
Yanping Fu ◽  
Xiaomin Han ◽  
...  
Keyword(s):  
Gene Expression ◽  
Internal Control ◽  
Abiotic Stresses ◽  
Target Genes ◽  
Expression Patterns ◽  
Control Measures ◽  
Rapid Expansion ◽  
Pcr Analysis ◽  
Data Normalization ◽  
Qrt Pcr

BackgroundStellera chamaejasmeLinn, an important poisonous plant of the China grassland, is toxic to humans and livestock. The rapid expansion ofS. chamaejasmehas greatly damaged the grassland ecology and, consequently, seriously endangered the development of animal husbandry. To draft efficient prevention and control measures, it has become more urgent to carry out research on its adaptive and expansion mechanisms in different unfavorable habitats at the genetic level. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used technique for studying gene expression at the transcript level; however, qRT-PCR requires reference genes (RGs) as endogenous controls for data normalization and only through appropriate RG selection and qRT-PCR can we guarantee the reliability and robustness of expression studies and RNA-seq data analysis. Unfortunately, little research on the selection of RGs for gene expression data normalization inS. chamaejasmehas been reported.MethodIn this study, 10 candidate RGs namely,18S,60S,CYP,GAPCP1,GAPDH2,EF1B,MDH,SAND,TUA1, andTUA6, were singled out from the transcriptome database ofS. chamaejasme, and their expression stability under three abiotic stresses (drought, cold, and salt) and three hormone treatments (abscisic acid, ABA; gibberellin, GA; ethephon, ETH) were estimated with the programs geNorm, NormFinder, and BestKeeper.ResultOur results showed thatGAPCP1andEF1Bwere the best combination for the three abiotic stresses, whereasTUA6andSAND,TUA1andCYP,GAPDH2and60Swere the best choices for ABA, GA, and ETH treatment, respectively. Moreover,GAPCP1and60Swere assessed to be the best combination for all samples, and18Swas the least stable RG for use as an internal control in all of the experimental subsets. The expression patterns of two target genes (P5CS2andGI) further verified that the RGs that we selected were suitable for gene expression normalization.DiscussionThis work is the first attempt to comprehensively estimate the stability of RGs inS. chamaejasme. Our results provide suitable RGs for high-precision normalization in qRT-PCR analysis, thereby making it more convenient to analyze gene expression under these experimental conditions.

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

2018 ◽  
Vol 20 (1) ◽  
pp. 34 ◽  
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.

scholarly journals Selection of Reference Genes for qRT-PCR Analysis of Gene Expression in Stipa grandis during Environmental Stresses

PLoS ONE ◽  
2017 ◽  
Vol 12 (1) ◽  
pp. e0169465 ◽  
Author(s):  
Dongli Wan ◽  
Yongqing Wan ◽  
Qi Yang ◽  
Bo Zou ◽  
Weibo Ren ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Environmental Stresses ◽  
Pcr Analysis ◽  
Qrt Pcr ◽  
Selection Of

Reference gene selection and evaluation for expression analysis using qRT-PCR in Galeruca daurica (Joannis)

2016 ◽  
Vol 107 (3) ◽  
pp. 359-368 ◽  
Author(s):  
Y. Tan ◽  
X.-R. Zhou ◽  
B.-P. Pang
Keyword(s):  
Reference Gene ◽  
Reference Genes ◽  
Target Genes ◽  
Gene Selection ◽  
Developmental Stages ◽  
Pcr Analysis ◽  
Experimental Conditions ◽  
Reference Gene Selection ◽  
Functional Studies ◽  
Qrt Pcr

AbstractQuantitative real-time PCR (qRT-PCR) has been used extensively to analyze gene expression and decipher gene function. To obtain the optimal and stable normalization factors for qRT-PCR, selection and validation of reference genes should be conducted in diverse conditions. In insects, more and more studies confirmed the necessity and importance of reference gene selection. In this study, eight traditionally used reference genes in Galeruca daurica (Joannis) were assessed, using qRT-PCR, for suitability as normalization genes under different experimental conditions using four statistical programs: geNorm, Normfinder, BestKeeper and the comparative ΔCt method. The genes were ranked from the most stable to the least stable using RefFinder. The optimal suite of recommended reference genes was as follows: succinate dehydrogenase (SDHA) and tubulin-alpha (TUB-α) for temperature-treated larvae; ribosomal protein L32, SDHA and glutathione S-transferase were best for all developmental stages; ACT and TUB-α for male and female adults; SDHA and TUB-α were relatively stable and expressed in different tissues, both diapause and non-diapause adults. Reference gene evaluation was validated using expression of two target genes: the P450 CYP6 gene and the heat shock protein gene Hsp70. These results confirm the importance of custom reference gene selection when studies are conducted under diverse experimental conditions. A standardized qRT-PCR analysis procedure for gene functional studies is provided that could be useful in studies on other insect species.

scholarly journals Selection of Reference Genes for qRT-PCR in Bletilla striata under Heat Stress

2021 ◽  
Vol 25 (03) ◽  
pp. 547-554
Author(s):  
Fang Liang
Keyword(s):  
Internal Control ◽  
Reference Genes ◽  
Elongation Factor ◽  
Pcr Analysis ◽  
Bletilla Striata ◽  
Traditional Chinese Herbal Medicine ◽  
Qrt Pcr ◽  
Wide Range ◽  
Root Tuber ◽  
Different Tissues

Bletilla striata (Thunb.) Reihb.f., a traditional Chinese herbal medicine, has attracted increasing attention because of its wide range of applicability to the medical field and chemical industry. B. striata has been identified to be particularly sensitive to high temperatures. Thus, the study of temperature stress on gene transcription is of interest in the field. Use of reliable reference genes is essential for qRT-PCR analysis of genes. However, little information regarding suitable reference genes for the genus Bletilla has been published. In this study, the sequences of seven potential reference genes in B. striata were obtained via a homology cloning strategy. These genes were as follows: glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18S), elongation factor 1 alpha (EF1α), α-tubulin (TUA), β-tubulin (TUB), ubiquitin (UBI), and NAC domain protein (NAC). We then evaluated the stability levels of these transcripts in different tissues (root, tuber, and leaf) exposed to high temperature using three conventional software and comprehensive RefFinder algorithms. The results indicated that 18S and TUB were the best internal control genes among different periods of heat treatment and that a combination of 18S and UBI was the best in different tissues. Altogether, 18S and UBI were identified to be the best reference genes for all the samples, while NAC and TUA were the least stable reference genes. The results will be useful for studies on target gene expression in plants of the Bletilla genus. © 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers © 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers©

scholarly journals Reference genes for qRT-PCR normalisation in different tissues, developmental stages, and stress conditions of Hypericum perforatum

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7133 ◽  
Author(s):  
Wen Zhou ◽  
Shiqiang Wang ◽  
Lei Yang ◽  
Yan Sun ◽  
Qian Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candidate Genes ◽  
Reference Genes ◽  
Hypericum Perforatum ◽  
Developmental Stages ◽  
Pcr Analysis ◽  
Potential Candidate ◽  
Qrt Pcr ◽  
Expression Studies ◽  
Different Tissues

Hypericum perforatum L. is a widely known medicinal herb used mostly as a remedy for depression because it contains high levels of naphthodianthrones, phloroglucinols, alkaloids, and some other secondary metabolites. Quantitative real-time PCR (qRT-PCR) is an optimized method for the efficient and reliable quantification of gene expression studies. In general, reference genes are used in qRT-PCR analysis because of their known or suspected housekeeping roles. However, their expression level cannot be assumed to remain stable under all possible experimental conditions. Thus, the identification of high quality reference genes is essential for the interpretation of qRT-PCR data. In this study, we investigated the expression of 14 candidate genes, including nine housekeeping genes (HKGs) (ACT2, ACT3, ACT7, CYP1, EF1-α, GAPDH, TUB-α, TUB-β, and UBC2) and five potential candidate genes (GSA, PKS1, PP2A, RPL13, and SAND). Three programs—GeNorm, NormFinder, and BestKeeper—were applied to evaluate the gene expression stability across four different plant tissues, four developmental stages and a set of abiotic stress and hormonal treatments. Integrating all of the algorithms and evaluations revealed that ACT2 and TUB-β were the most stable combination in different developmental stages samples and all of the experimental samples. ACT2, TUB-β, and EF1-α were identified as the three most applicable reference genes in different tissues and stress-treated samples. The majority of the conventional HKGs performed better than the potential reference genes. The obtained results will aid in improving the credibility of the standardization and quantification of transcription levels in future expression studies on H. perforatum.

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.

scholarly journals Selection and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis in Needles of Larix olgensis under Abiotic Stresses

Forests ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 193
Author(s):  
Dandan Li ◽  
Sen Yu ◽  
Minzhen Zeng ◽  
Xiao Liu ◽  
Jia Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Real Time ◽  
Reference Genes ◽  
Pcr Analysis ◽  
Stable Gene ◽  
Larix Olgensis ◽  
Qrt Pcr ◽  
Study Gene Expression ◽  
Selection Of

Larix olgensis Henry is an important afforestation species in northeastern China because of its fast juvenile growth, high-quality timber, and significant economic and ecological values. The selection of appropriate reference genes is necessary for the normalization of gene expression determination during quantitative real-time polymerase chain reaction (qRT-PCR) experiments. In this study, qRT-PCR was used to study gene expression. Three software packages geNorm, NormFinder, BestKeeper were used, and a comprehensive ranking of candidate reference genes was produced based on their output to evaluate the expression stability of 16 candidate reference genes from L. olgensis under drought, salt, cold, and heat stress. PP2A-1 and GAPDH ranked as the most stable reference genes under drought and cold stress, PP2A-1 and UBQ10 were most stable under salt stress, and TIP41 and ACT2 were most stable under heat stress. The least stable gene was ADP, which ranked the last under all treatments. Expression profile analysis of the antioxidant gene CAT using the two most stable and the single least stable reference genes under each stress further verified that the selected reference genes were suitable for gene expression normalization. This study provides an important foundation for the selection of suitable reference genes for the normalization and quantification of L. olgensis gene expression under abiotic stress conditions.

scholarly journals Identification of stable reference genes for lipopolysaccharide-stimulated macrophage gene expression studies

2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Roshini Kalagara ◽  
Weimin Gao ◽  
Honor L. Glenn ◽  
Colleen Ziegler ◽  
Laura Belmont ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ribosomal Protein ◽  
Reference Gene ◽  
Reference Genes ◽  
Target Genes ◽  
Stable Gene ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Expression Studies ◽  
Gene Expression Studies

Gene expression studies which utilize lipopolysaccharide (LPS)-stimulated macrophages to model immune signaling are widely used for elucidating the mechanisms of inflammation-related disease. When expression levels of target genes are quantified using Real-Time quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), they are analyzed in comparison to reference genes, which should have stable expression. Judicious selection of reference genes is, therefore, critical to interpretation of qRT-PCR results. Ideal reference genes must be identified for each experimental system and demonstrated to remain constant under the experimental conditions. In this study, we evaluated the stability of eight common reference genes: Beta-2-microglobulin (B2M), Cyclophilin A/Peptidylprolyl isomerase A, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), Hypoxanthine Phosphoribosyltransferase 1, Large Ribosomal Protein P0, TATA box binding protein, Ubiquitin C (UBC), and Ribosomal protein L13A. Expression stability of each gene was tested under different conditions of LPS stimulation and compared to untreated controls. Reference gene stabilities were analyzed using Ct value comparison, NormFinder, and geNorm. We found that UBC, closely followed by B2M, is the most stable gene, while the commonly used reference gene GAPDH is the least stable. Thus, for improved accuracy in evaluating gene expression levels, we propose the use of UBC to normalize PCR data from LPS-stimulated macrophages.

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