scholarly journals Selection of appropriate reference genes for RT-qPCR analysis under abiotic stress and hormone treatment in celery

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7925 ◽  
Author(s):  
Kai Feng ◽  
Jie-xia Liu ◽  
Guo-Ming Xing ◽  
Sheng Sun ◽  
Sen Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Abiotic Stress ◽  
Reference Gene ◽  
Reference Genes ◽  
Regulatory Mechanism ◽  
Biological Response ◽  
Hormone Treatment ◽  
Yield And Quality ◽  
Qpcr Analysis ◽  
Selection Of

Celery is one of the most important vegetable crop and its yield and quality is influenced by many environmental factors. Researches on gene expression not only help to unravel the molecular regulatory mechanism but also identify the key genes in the biological response. RT-qPCR is a commonly used technology to quantify the gene expression. Selecting an appropriate reference gene is an effective approach to improve the accuracy of RT-qPCR assay. To our knowledge, the evaluation of reference genes under different treatments in celery has not been reported yet. In this study, the expression stabilities of eight candidate reference genes (ACTIN, eIF-4α, GAPDH, TBP, TUB-A, UBC, TUB-B, and EF-1α) under abiotic stresses (heat, cold, drought, and salt) and hormone treatments (SA, MeJA, GA, and ABA) were detected. The expression stabilities of candidate genes were compared and ranked by geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder programs. The results calculated by different programs were not completely consistent. Considering the comprehensive analysis results, ACTIN was the most stable reference gene and TUB-B showed the worst expression stabilities under the selected abiotic stress and hormone treatments in celery. The reliability of reference genes was further confirmed by the normalization of CAT1 gene under drought stress. This study presented evidences and basis to select the appropriate reference genes under different treatments in celery.

scholarly journals Validation of the Reference Genes for the Gene Expression Studies in Chicken DT40 Cell Line

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 372
Author(s):  
Aleksandra Dunislawska ◽  
Anna Slawinska ◽  
Maria Siwek
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Relative Gene Expression ◽  
Dt40 Cell ◽  
Relative Gene ◽  
Chicken Dt40 Cell ◽  
Selection Of

The selection of a suitable reference gene assures a reliable gene expression analysis when using the qPCR method. Normalization of the reaction is based on the basic metabolism genes. These genes show a constant, unregulated expression in all cells and function throughout their lifetime. In the current study, seven reference gene candidates were screened using RT-qPCR, to determine the best-matched pair of reference genes in the chicken DT40 cell line. The DT40 was derived from bursal lymphoma cells that were subjected to RAV-1 bird retroviral infection. It is a simplified in vitro model that allows tracking the direct interaction of stimulants on the lymphoid population and profiling of the hepatocellular B cell transcriptome. The reference gene analysis was carried out using statistical tools integrating four independent methods—geNorm, Best Keeper, NormFinder, delta Ct and RefFinder. Based on the selected reference genes, the relative gene expression analysis was done using the ddCt method. Complete relative gene expression study on a panel of the target genes revealed that proper selection of reference genes depending on the tissue eliminate decreases in data quality. The SDHA and RPL4 genes constitute stable internal controls as reference genes when analyzing gene expression in the DT40 cell line.

scholarly journals Expression Stability of Internal Reference Gene in Response to Trichoderma polysporum Infection in Avena fatua L.

2021 ◽  
Author(s):  
Haixia Zhu ◽  
Yongqiang Ma ◽  
Liang Cheng
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Expression Analysis ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Avena Fatua ◽  
Expression Stability ◽  
Internal Reference ◽  
Qpcr Analysis ◽  
Suitable Combination

Abstract In order to construct a RT-qPCR system suitable for response of Avena fatua L. to Trichoderma polysporum , and screen stable internal reference genes, GeNorm, NormFinder, BestKeeper and RefFinde were used to perform SYBR Green-based RT-qPCR analysis on 8 candidate internal reference genes ( 18S , 28S , TUA , UBC , ACT , GAPDH , TBP and EF-1 ) in A. fatua samples after inoculation of T. polysporum Strain HZ-31. The results showed that TBP , 18S and UBC were the most stable internal reference genes, TBP and TUA , TBP and GAPDH , 18S and TBP , UBC and 18S were the most suitable combination of the two internal reference genes, which could be used as the internal reference genes for functional gene expression analysis during the interaction between T. polysporum and A. fatua .

scholarly journals Selection of reliable reference genes for RT-qPCR analysis during developmental stages and abiotic stress in Setaria viridis

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Polyana Kelly Martins ◽  
Valéria Mafra ◽  
Wagner Rodrigo de Souza ◽  
Ana Paula Ribeiro ◽  
Felipe Vinecky ◽  
...  
Keyword(s):  
Abiotic Stress ◽  
Reference Genes ◽  
Developmental Stages ◽  
Setaria Viridis ◽  
Qpcr Analysis ◽  
Selection Of

scholarly journals Identification of Suitable Reference Genes for Expression Studies in Pomegranate Under Different Biotic and Abiotic Stress Conditions

2021 ◽  
Author(s):  
Puspha Doddaraju ◽  
Pavan Kumar ◽  
Mahesh S Dashyal ◽  
Manjunath Girigowda
Keyword(s):  
Gene Expression ◽  
Abiotic Stress ◽  
Reference Gene ◽  
Reference Genes ◽  
Bacterial Blight ◽  
Candidate Reference Gene ◽  
Target Gene ◽  
Stress Conditions ◽  
Biotic And Abiotic Stress ◽  
Expression Studies

Abstract Pomegranate (Punica granatum) is an important economic fruit crop, facing many biotic and abiotic challenges during cultivation. Several research programs are in progress to understand both biotic and abiotic stress factors and mitigate these challenges using gene expression studies based on the qPCR approach. However, research publications are not available yet to select the standard reference gene for normalizing target gene expression values in pomegranate. The most suitable candidate reference gene is required to ensure precise and reliable results for qPCR analysis. In the current research, eight candidate reference genes' stability was evaluated under different stress conditions using different algorithms such as ∆Ct, geNorm, BestKeeper, NormFinder and RefFinder. The various algorithms revealed that EFA1 and 18S rRNA were common and most stable reference genes (RGs) under abiotic and wilt stress. Whereas comprehensive ranking by RefFinder showed GAPDH and CYPF were the most stable RGs under combined biotic (pooled samples of all biotic stress) and bacterial blight samples. The two most stable reference genes are adequate for normalizing target gene expression under wilt, nematode, bacterial blight and abiotic stress conditions using qPCR. The above data provide comprehensive details for the selection of a candidate reference gene in various stresses in pomegranate

132 REFERENCE GENE SCREENING FOR ANALYZING GENE EXPRESSION ACROSS FEMALE GOAT TISSUE

2014 ◽  
Vol 26 (1) ◽  
pp. 179
Author(s):  
X.-D. Zhang ◽  
Y. Zhang ◽  
X. Liu ◽  
Y.-S. Li ◽  
J.-P. Ding ◽  
...  
Keyword(s):  
Gene Expression ◽  
Small Intestine ◽  
Reference Gene ◽  
Reference Genes ◽  
Absolute Quantification ◽  
Expression Levels ◽  
Boer Goat ◽  
Standard Curve ◽  
The Stability ◽  
Selection Of

Quantitative RT-PCR (RT-qPCR) is an important method for investigating changes in mRNA expression levels in cells and tissues. Selection of the proper reference genes is very important when calibrating the results of RT-qPCR. Studies on the selection of reference genes in goat tissues are limited, despite the economic importance of their meat and dairy products. We applied absolute quantification using the standard curve method to the detection of the expression levels of 8 reference gene candidates (18S, TBP, YWHAZ, HMBS, ACTB, HPRT1, GAPDH, and EEF1A2) in 10 different tissue types (heart, liver, spleen, lung, kidney, stomach, uterus, ovary, small intestine, and muscle) sourced from a 5-month-old female Boer goat. All the experiments were performed using 3 biological replicates and technical triplicates of each cDNA sample. The optimal reference gene combination was selected according to the results determined by geNorm, NormFinder, and Bestkeeper software packages. The analyses showed that tissue is an important variability factor in gene expression stability. When all tissues were considered, 18S, TBP, and HMBS with the smallest paired variation coefficient is the optimal reference combination for calibrating RT-qPCR analysis of gene expression from goat tissues. The stability of each gene was evaluated, taking into account all of the data. The stability values (M) for these genes were 0.643/0.634/0.799, 0.156/0.467/0.584, and 0.396/0.445/0.566 by geNorm, NormFinder, and Bestkeeper, respectively. Dividing the dataset by different tissues, ACTB was the most stable in stomach, small intestine, and ovary, 18S in heart and spleen, HMBS in uterus and lung, TBP in liver, HPRT1 in kidney, and GAPDH in muscle. Overall, this study provided valuable information about the female goat reference genes that can be used in order to perform a proper normalisation when relative quantification by RT-qPCR studies is undertaken. This work was supported by National ‘863’ Program (2011AA100307-4), the Technology Innovation Project – Special Program and the Science and Technology Program of Anhui Province (11Z0101095 and 11010302108).

scholarly journals Selection of reference genes for quantitative real-time PCR analysis in halophytic plantRhizophora apiculata

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5226 ◽  
Author(s):  
Ankush Ashok Saddhe ◽  
Manali Ramakant Malvankar ◽  
Kundan Kumar
Keyword(s):  
Gene Expression ◽  
Salt Stress ◽  
Real Time ◽  
Reference Gene ◽  
Reference Genes ◽  
Candidate Reference Gene ◽  
Expression Level ◽  
Bisphosphate Carboxylase ◽  
Qrt Pcr ◽  
Selection Of

Rhizophora apiculatais a halophytic, small mangrove tree distributed along the coastal regions of the tropical and subtropical areas of the world. They are natural genetic reservoirs of salt adaptation genes and offer a unique system to explore adaptive mechanisms under salinity stress. However, there are no reliable studies available on selection and validation of reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) inR. apiculataphysiological tissues and in salt stress conditions. The selection of appropriate candidate reference gene for normalization of qRT-PCR data is a crucial step towards relative analysis of gene expression. In the current study, seven genes such as elongation factor 1α (EF1α), Ubiquitin (UBQ), β-tubulin (β-TUB), Actin (ACT), Ribulose1,5-bisphosphate carboxylase/oxygenase (rbcL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 18S rRNA (18S) were selected and analyzed for their expression stability. Physiological tissues such as leaf, root, stem, and flower along with salt stress leaf samples were used for selection of candidate reference genes. The high-quality expression data was obtained from biological replicates and further analyzed using five different programs such as geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder. All algorithms comprehensively rankedEF1α followed byACTas the most stable candidate reference genes inR. apiculataphysiological tissues. Moreover, β-TUBand 18S were ranked as moderately stable candidate reference genes, while GAPDH andrbcLwere least stable reference genes. Under salt stress,EF1α was comprehensively recommended top-ranked candidate reference gene followed byACTand 18S. In order to validate the identified most stable candidate reference genes,EF1α,ACT, 18S andUBQwere used for relative gene expression level of sodium/proton antiporter (NHX) gene under salt stress. The expression level ofNHXvaried according to the internal control which showed the importance of selection of appropriate reference gene. Taken together, this is the first ever systematic attempt of selection and validation of reference gene for qRT-PCR inR. apiculataphysiological tissues and in salt stress. This study would promote gene expression profiling of salt stress tolerance related genes inR. apiculata.

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