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

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
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.

scholarly journals Selecting and validating reference genes for quantitative real-time PCR in Plutella xylostella (L.)

Genome ◽  
2018 ◽  
Vol 61 (5) ◽  
pp. 349-358 ◽  
Author(s):  
Yanchun You ◽  
Miao Xie ◽  
Liette Vasseur ◽  
Minsheng You
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Plutella Xylostella ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Developmental Stages ◽  
Quantitative Real Time Pcr ◽  
Experimental Conditions ◽  
Expression Studies ◽  
Gene Expression Studies

Gene expression analysis provides important clues regarding gene functions, and quantitative real-time PCR (qRT-PCR) is a widely used method in gene expression studies. Reference genes are essential for normalizing and accurately assessing gene expression. In the present study, 16 candidate reference genes (ACTB, CyPA, EF1-α, GAPDH, HSP90, NDPk, RPL13a, RPL18, RPL19, RPL32, RPL4, RPL8, RPS13, RPS4, α-TUB, and β-TUB) from Plutella xylostella were selected to evaluate gene expression stability across different experimental conditions using five statistical algorithms (geNorm, NormFinder, Delta Ct, BestKeeper, and RefFinder). The results suggest that different reference genes or combinations of reference genes are suitable for normalization in gene expression studies of P. xylostella according to the different developmental stages, strains, tissues, and insecticide treatments. Based on the given experimental sets, the most stable reference genes were RPS4 across different developmental stages, RPL8 across different strains and tissues, and EF1-α across different insecticide treatments. A comprehensive and systematic assessment of potential reference genes for gene expression normalization is essential for post-genomic functional research in P. xylostella, a notorious pest with worldwide distribution and a high capacity to adapt and develop resistance to insecticides.

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 Selection and validation of reference genes for measuring gene expression in Toona ciliata under different experimental conditions by quantitative real-time PCR analysis

2020 ◽  
Author(s):  
Huiyun Song ◽  
Wenmai Mao ◽  
Zhihao Duan ◽  
Qingmin Que ◽  
Wei Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Accurate Method ◽  
Pcr Analysis ◽  
Quantitative Real Time Pcr ◽  
Experimental Conditions ◽  
Toona Ciliata

Abstract Background:Before studying gene expression of different organisms, it is important to determine the best reference gene. At present, the most accurate method of detecting gene expression is quantitative real-time PCR (RT-qPCR). With this method, reference genes that are stable in different biological systems and under different conditions can be obtained. Toona ciliata Roem ( T. ciliata ). is a valuable and fast-growing timber specie. In this study, 20 reference genes were identified using RT-qPCR, as a primary prerequisite for future gene expression analysis. Four different methods, geNorm, NormFinder, BestKeeper, and RankAggreg were used to evaluate the expression stability of the 20 candidate reference genes in various tissues under different conditions.Results:The experimental results showed that TUB-α was the most stably expressed reference gene across all samples and UBC17 was the most stable in leaves and young stems under Hypsipyla robusta ( H. robusta ) and methyl jasmonate (MeJA) treatments. In addition, PP2C59 and UBC5B were the best-performing genes in leaves under H. robusta treatment, while HIS1 and ACT7 were the best reference genes in young stems. The two best reference genes were 60S-18 and TUB-α after treatment at 4 °C. The expression of HIS6 and MUB1 was the most stable under PEG6000 treatment. The accuracy of the selected reference genes was verified using the transcription factor MYB3 ( TcMYB3) gene.Conclusions:This is the first report to verify the best reference genes for normalizing gene expression in T. ciliata under different conditions, which will facilitate future elucidation of gene regulations in this species.

scholarly journals Systematic selection and validation of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Nitraria tangutorum

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Bo Wang ◽  
Huirong Duan ◽  
Peifang Chong ◽  
Shiping Su ◽  
Lishan Shan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Expression Stability ◽  
Quantitative Real Time Pcr ◽  
Experimental Conditions ◽  
Nitraria Tangutorum ◽  
Multiple Species ◽  
Suitable Reference

Abstract Suitable reference genes can be used to calibrate the error in quantitative real-time PCR (qPCR) experiments, making the results more credible. However, there are no reference genes suitable for multiple species and under different experimental conditions. Nitraria tangutorum Bobr. is a typical plant native to desert areas. It is drought-resistant, saline-alkali resistant, extreme temperatures-resistant, and has strong adaptability. To date, the importance of this germplasm has not been sufficiently understood; therefore, it is still unclear which genes can be used as reference genes to calibrate qPCR data of N. tangutorum. In this study we analyzed the expression levels of 10 candidate reference genes (ACT, GAPDH, TUA, TUB, CYP, UBC, His, PP2A, HSP, and EF1-α) in N. tangutorum seedlings under a series of experimental conditions, including in different organs (root, stem, and leaf) and under abiotic stresses (salt, drought, heat, and cold) and hormone stimuli (abscisic acid) by qPCR. Three software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate the expression stability of the ten genes. Comprehensive analysis showed that EF1-α and His had the best expression stability, whereas HSP was the least suitable as a reference gene. The expression profile of NtCER7, a gene related to the regulation of cuticular wax biosynthesis in N. tangutorum, verified the accuracy of the experimental results. Based on this study, we recommend EF1-α and His as suitable reference genes for N. tangutorum. This paper provides the first data on stable reference genes in N. tangutorum, which will be beneficial to studying the gene expression of N. tangutorum and other Nitraria species in the future.

scholarly journals Reference Gene Selection for qPCR Normalization ofKosteletzkya virginicaunder Salt Stress

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
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.

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

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.

scholarly journals Selection and validation of reference genes for measuring gene expression in Toona ciliata under different experimental conditions by quantitative real-time PCR analysis

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Huiyun Song ◽  
Wenmai Mao ◽  
Zhihao Duan ◽  
Qingmin Que ◽  
Wei Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Accurate Method ◽  
Pcr Analysis ◽  
Quantitative Real Time Pcr ◽  
Experimental Conditions ◽  
Toona Ciliata

Abstract Background Before studying gene expression of different organisms, it is important to determine the best reference gene. At present, the most accurate method of detecting gene expression is quantitative real-time PCR (RT-qPCR). With this method, reference genes that are stable in different biological systems and under different conditions can be obtained. Toona ciliata Roem (T. ciliata). is a valuable and fast-growing timber specie. In this study, 20 reference genes were identified using RT-qPCR, as a primary prerequisite for future gene expression analysis. Four different methods, geNorm, NormFinder, BestKeeper, and RankAggreg were used to evaluate the expression stability of the 20 candidate reference genes in various tissues under different conditions. Results The experimental results showed that TUB-α was the most stably expressed reference gene across all samples and UBC17 was the most stable in leaves and young stems under Hypsipyla robusta (H. robusta) and methyl jasmonate (MeJA) treatments. In addition, PP2C59 and UBC5B were the best-performing genes in leaves under H. robusta treatment, while HIS1 and ACT7 were the best reference genes in young stems. The two best reference genes were 60S-18 and TUB-α after treatment at 4 °C. The expression of HIS6 and MUB1 was the most stable under PEG6000 treatment. The accuracy of the selected reference genes was verified using the transcription factor MYB3 (TcMYB3) gene. Conclusions This is the first report to verify the best reference genes for normalizing gene expression in T. ciliata under different conditions, which will facilitate future elucidation of gene regulations in this species.

scholarly journals Selection and validation of reference genes for measuring gene expression in Toona ciliata under different experimental conditions by quantitative real-time PCR analysis

2020 ◽  
Author(s):  
Huiyun song ◽  
Wenmai Mao ◽  
Zhihao Duan ◽  
Qingmin Que ◽  
Wei Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Accurate Method ◽  
Pcr Analysis ◽  
Quantitative Real Time Pcr ◽  
Experimental Conditions ◽  
Toona Ciliata

Abstract Background: Before studying gene expression of different organisms, it is important to determine the best reference gene. At present, the most accurate method of detecting gene expression is quantitative real-time PCR (RT-qPCR). With this method, reference genes that are stable in different biological systems and under different conditions can be obtained. Toona ciliata Roem (T. ciliata). is a valuable and fast-growing timber specie. In this study, 20 reference genes were identified using RT-qPCR, as a primary prerequisite for future gene expression analysis. Four different methods, geNorm, NormFinder, BestKeeper, and RankAggreg were used to evaluate the expression stability of the 20 candidate reference genes in various tissues under different conditions. Results: The experimental results showed that TUB-α was the most stably expressed reference gene across all samples and UBC17 was the most stable in leaves and young stems under Hypsipyla robusta (H. robusta) and methyl jasmonate (MeJA) treatments. In addition, PP2C59 and UBC5B were the best-performing genes in leaves under H. robusta treatment, while HIS1 and ACT7 were the best reference genes in young stems. The two best reference genes were 60S-18 and TUB-α after treatment at 4 °C. The expression of HIS6 and MUB1 was the most stable under PEG6000 treatment. The accuracy of the selected reference genes was verified using the transcription factor MYB3 (TcMYB3) gene. Conclusions: This is the first report to verify the best reference genes for normalizing gene expression in T. ciliata under different conditions, which will facilitate future elucidation of gene regulations in this species.

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