scholarly journals Selection of reliable reference genes for quantitative RT-PCR in garlic under salt stress

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7319 ◽  
Author(s):  
Guanglong Wang ◽  
Chang Tian ◽  
Yunpeng Wang ◽  
Faxiang Wan ◽  
Laibao Hu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Salt Stress ◽  
Reference Genes ◽  
Expression Profiles ◽  
Accurate Determination ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Specific Reference ◽  
Variable Expression ◽  
Selection Of

Quantitative real-time reverse-transcriptase PCR (qRT-PCR) has been frequently used for detecting gene expression. To obtain reliable results, selection of suitable reference genes is a fundamental and necessary step. Garlic (Allium sativum), a member from Alliaceae family, has been used both as a food flavoring and as a traditional medicine. In the present study, garlic plants were exposed to salt stress (200 mM NaCl) for 0, 1, 4 and 12 h, and garlic roots, bulbs, and leaves were harvested for subsequent analysis. The expression stability of eight candidate reference genes, eukaryotic translation initiation factor 4α (eIF-4α), actin (ACTIN), tubulin β-7 (TUB7), TAP42-interacting protein of 41 kDa (TIP41), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), SAND family protein (SAND), elongation factor 1 alpha (EF-1α), and protein phosphatase 2A (PP2A) were evaluated by geNorm, NormFinder, and BestKeeper. All genes tested displayed variable expression profiles under salt stress. In the leaf and root group, ACTIN was the best reference gene for normalizing gene expression. In garlic clove, ACTIN and SAND were the least variable, and were suitable for gene expression studies under salt stress; these two genes also performed well in all samples tested. Based on our results, we recommend that it is essential to use specific reference genes in different situations to obtain accurate results. Using a combination of multiple stable reference genes, such as ACTIN and SAND, to normalize gene expression is encouraged. The results from the study will be beneficial for accurate determination of gene expression in garlic and other plants.

scholarly journals Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization inIris. lacteavar.chinensisRoots under Cadmium, Lead, and Salt Stress Conditions

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Chun-Sun Gu ◽  
Liang-qin Liu ◽  
Chen Xu ◽  
Yan-hai Zhao ◽  
Xu-dong Zhu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Salt Stress ◽  
Real Time ◽  
Reference Genes ◽  
Elongation Factor ◽  
Stress Conditions ◽  
Translation Initiation Factor ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Wide Range

Quantitative real time PCR (RT-qPCR) has emerged as an accurate and sensitive method to measure the gene expression. However, obtaining reliable result depends on the selection of reference genes which normalize differences among samples. In this study, we assessed the expression stability of seven reference genes, namely, ubiquitin-protein ligase UBC9 (UBC), tubulin alpha-5 (TUBLIN), eukaryotic translation initiation factor (EIF-5A), translation elongation factor EF1A (EF1α), translation elongation factor EF1B (EF1b), actin11 (ACTIN), and histone H3 (HIS), inIris. lacteavar.chinensis(I. lacteavar.chinensis) root when the plants were subjected to cadmium (Cd), lead (Pb), and salt stress conditions. All seven reference genes showed a relatively wide range of threshold cycles (Ct) values in different samples. GeNorm and NormFinder algorithms were used to assess the suitable reference genes. The results from the two software units showed thatEIF-5AandUBCwere the most stable reference genes across all of the tested samples, whileTUBLINwas unsuitable as internal controls.I. lacteavar.chinensisis tolerant to Cd, Pb, and salt. Our results will benefit future research on gene expression in response to the three abiotic stresses.

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 Selection of Reference Genes for qRT-PCR Analysis in Medicinal Plant Glycyrrhiza under Abiotic Stresses and Hormonal Treatments

Plants ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 1441
Author(s):  
Yuping Li ◽  
Xiaoju Liang ◽  
Xuguo Zhou ◽  
Zhigeng Wu ◽  
Ling Yuan ◽  
...  
Keyword(s):  
Abiotic Stresses ◽  
Reference Genes ◽  
Citrate Synthase ◽  
Expression Profiles ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Pcr Analysis ◽  
Congeneric Species ◽  
Qrt Pcr ◽  
Hormonal Treatments

Best known as licorice, Glycyrrhiza Linn., a genus of herbaceous perennial legume, has been used as a traditional herbal medicine in Asia and a flavoring agent for tobacco and food industry in Europe and America. Abiotic stresses and hormonal treatments can significantly impact the development and metabolism of secondary metabolites in Glycyrrhiza. To better understand the biosynthesis of the trace-amount bioactive compounds, we first screened for the suitable reference genes for quantitative real-time reverse transcription PCR (qRT-PCR) analysis in Glycyrrhiza. The expression profiles of 14 candidate reference genes, including Actin1 (ACT), Clathrin complex AP1 (CAC), Cyclophilin (CYP), Heat-shock protein 40 (DNAJ), Dehydration responsive element binding gene (DREB), Translation elongation factor1 (EF1), Ras related protein (RAN), Translation initiation factor (TIF1), β-Tubulin (TUB), Ubiquitin-conjugating enzyme E2 (UBC2), ATP binding-box transpoter 2 (ABCC2), COP9 signal compex subunit 3 (COPS3), Citrate synthase (CS), and R3H domain protein 2 (R3HDM2) from two congeneric species, Glycyrrhiza uralensis F. and Glycyrrhiza inflata B., were examined under abiotic stresses (osmotic and salinity) and hormonal treatments (Abscisic acid (ABA) and methyl jasmonic acid (MeJA)) using a panel of software, including geNorm, NormFinder, BestKeeper, and Delta CT. The overall stability, however, was provided by RefFinder, a comprehensive ranking system integrating inputs from all four algorithms. In G. uralensis, the most stable reference genes under osmotic stress, salt stress, ABA treatment, and MeJA treatment were TIF1, DNAJ, CS, and ABCC2 for leaves and DNAJ, DREB, CAC, and CAC for roots, respectively. In comparison, the top ranked genes were TUB, CAC, UBC2, and RAN for leaves and TIF1, ABCC2, CAC, and UBC2 for roots, respectively, under stress and hormonal treatments in G. inflata. ACT and TIF1, on the other hand, were the least stable genes under the most experimental conditions in the two congeneric species. Finally, our survey of the reference genes in legume shows that EF, ACT, UBC2, and TUB were the top choices for the abiotic stresses while EF, UBC2, CAC, and ABCC2 were recommended for the hormonal treatments in Leguminosae. Our combined results provide reliable normalizers for accurate gene quantifications in Glycyrrhiza species, which will allow us to exploit its medicinal potential in general and antiviral activities in particular.

scholarly journals Interindividual and Interethnic Variation in Genomewide Gene Expression: Insights into the Biological Variation of Gene Expression and Clinical Implications

2009 ◽  
Vol 55 (4) ◽  
pp. 774-785 ◽  
Author(s):  
Harris P Y Fan ◽  
Chen Di Liao ◽  
Brenda Yan Fu ◽  
Linda C W Lam ◽  
Nelson L S Tang
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Expression Profiles ◽  
Disease Diagnosis ◽  
Reference Intervals ◽  
Biological Variation ◽  
Interindividual Variation ◽  
Specific Reference ◽  
Diagnosis And Prognosis ◽  
Biological Differences

Abstract Background: Analysis of gene expression in peripheral blood samples is increasingly being applied in biomarker studies of disease diagnosis and prognosis. Although knowledge of interindividual and interethnic variation in gene expression is required to set ethnicity-specific reference intervals and to select reference genes and preferred markers from a list of candidate genes, few studies have attempted to characterize such biological variation on a genomewide scale. Methods: The genomewide expression profiles of 11 355 transcripts expressed among 210 multiethnic individuals of the HapMap project were obtained and analyzed; 4 replicates were included for each sample. The total biological CV in gene expression (CVb) was partitioned into interindividual (CVg), inter-ethnic group (CVe), and residual components by random-effects mixed models. Results: CVg was the major component of CVb, and the differences among transcripts were large (up to 38%). Distinct groups of genes were characterized by CV values and expression levels. Of the genes with lowest biological variation (CVb < 1.5%), 35 genes were highly expressed, whereas 32 had intermediate or low expression. Although CVg was almost always greater than CVe, we identified 10 genes in which ethnic variation predominated (range, 8%–18%). On the other hand, 17 annotated genes were highly variable with CVg values ranging between 15% and 38%. Conclusions: Genomewide analysis of gene expression variation demonstrated biological differences among transcripts. Transcripts with the least biological variation are better candidates for reference genes, whereas those with low interindividual variation may be good disease markers. The presence of interethnic variation suggests that ethnicity-specific reference intervals may be necessary.

scholarly journals Evaluation of Housekeeping Gene Expression Stability in Carnation (Dianthus caryophyllus)

2020 ◽  
Author(s):  
Huolin Luo ◽  
Wenjing Yu ◽  
Yuan Tao ◽  
Jonathan Hrovat ◽  
Ahui Xue ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Dianthus Caryophyllus ◽  
Housekeeping Gene ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Suitable Reference Gene ◽  
Expression Stability ◽  
Experimental Conditions

Abstract Background: The real-time quantitative reverse transcription PCR (RT-qPCR) is widely used for gene expression analysis, owing to its advantages of high specificity, sensitivity and repeatability. A suitable reference gene is an absolute prerequisite for accurate normalization, nevertheless, the frequently-used reference gene was reported unstable under different experimental conditions and causes failure to correctly analyze the expression of the interested gene. Therefore, it is vital to systematically evaluate the expression stability of these candidate reference genes before performing RT-qPCR. Results: In this study, two computational statistical methods were used, including geNorm and NormFinder, in order to determine the expression stability of 12 frequently-used reference genes in Dianthus caryophyllus across different experimental conditions. The results show that the expression stability of candidate genes varies greatly in different sample pools, which again proves the instability of these common housekeeping gene expressions. In general, the expression of UBQ10 (ubiquitin10), EF1a (elongation factor 1A) and TIP41 (TIP41-like family protein) were relatively stable under different experimental conditions, while the expression stability of 18S (18S Ribosome RNA), TIF5A (translation initiation factor 5A) and PP2A (protein phosphatase 2A) were relatively poor. Conclusion: EF1α, TIP41 and UBQ10 were considered the most appropriate reference genes when all samples were put together, while UBQ10 was most stable in exogenous hormone treatments. TUB and UBQ10 can be used as reliable internal control genes under stress, while CYP and TUA can act as reliable internal controls in different tissues. This is the first systematic study of selection of reference genes in Carnation, and will benefit future expression studies in this crop.

scholarly journals Evaluation of Candidate Reference Genes for Quantitative Gene Expression Studies in Tree Peony

2016 ◽  
Vol 141 (2) ◽  
pp. 99-111 ◽  
Author(s):  
Lin Zhou ◽  
Qianqian Shi ◽  
Yan Wang ◽  
Kui Li ◽  
Baoqiang Zheng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Ornamental Plants ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Expression Stability ◽  
Tree Peony ◽  
Different Tissues

Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) is a sensitive and widely used technique for gene expression analysis that depends on stability of the reference genes used for data normalization. Tree peony (Paeonia suffruticosa), known as one of the most famous traditional ornamental plants in China, is very popular in both domestic and international markets for its showy and colorful flowers. To date, no systematic studies on reference genes have been performed in tree peony with different flower colors. In this study, we evaluated the expression stability of 12 candidate reference genes in different tissues and five flower developmental stages of tree peony with six different colors by three algorithms: geNorm, NormFinder, and BestKeeper. The results showed that protein phosphatase 2A (PP2A), ubiquitin protein ligase (UPL), and ubiquitin (UBQ) were the most stable genes across all samples. Helicase, alpha-tubulin (TUA), and eukaryotic translation initiation factor 5A (EIF5A) also exhibited high expression stability in different tissues, in samples with different colors, and at different flower developmental stages. According to the geNorm analysis, the combination of two most stable reference genes was optimal for normalization in all tested sample sets in this study. To further validate the suitability of the reference genes identified in this study, the expression patterns of two putative homologs of chalcone synthase gene (PsCHS1) and chalcone isomerase gene (PsCHI1) were studied at different developmental stages of white flowers. The results provide information for transcriptional analyses in future studies of gene expression on tree peony flower development and pigmentation.

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.

scholarly journals Spatio-temporal selection of reference genes in the two congeneric species of Glycyrrhiza

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuping Li ◽  
Xiaoju Liang ◽  
Xuguo Zhou ◽  
Yu An ◽  
Ming Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Developmental Stage ◽  
Reference Genes ◽  
Developmental Stages ◽  
Individual Factors ◽  
Congeneric Species ◽  
Spatio Temporal ◽  
The Stability ◽  
Different Tissues ◽  
Selection Of

AbstractGlycyrrhiza, a genus of perennial medicinal herbs, has been traditionally used to treat human diseases, including respiratory disorders. Functional analysis of genes involved in the synthesis, accumulation, and degradation of bioactive compounds in these medicinal plants requires accurate measurement of their expression profiles. Reverse transcription quantitative real-time PCR (RT-qPCR) is a primary tool, which requires stably expressed reference genes to serve as the internal references to normalize the target gene expression. In this study, the stability of 14 candidate reference genes from the two congeneric species G. uralensis and G. inflata, including ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2, were evaluated across different tissues and throughout various developmental stages. More importantly, we investigated the impact of interactions between tissue and developmental stage on the performance of candidate reference genes. Four algorithms, including geNorm, NormFinder, BestKeeper, and Delta Ct, were used to analyze the expression stability and RefFinder, a comprehensive software, provided the final recommendation. Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual factors. In G. uralensis, the top three most stable reference genes across different tissues were R3HDM2, CAC and TUB, while CAC, CYP and ABCC2 were most suited for different developmental stages. CAC is the only candidate recommended for both biotic factors, which is reflected in the stability ranking for the spatio (tissue)-temporal (developmental stage) interactions (CAC, R3HDM2 and DNAJ). Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis. In summary, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential.

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