Validation of Reference Genes for RT-qPCR in Marine Bivalve Ecotoxicology: Systematic Review and Case Study

2016 ◽  
Author(s):  
Moritz Volland ◽  
Julián Blasco ◽  
Miriam Hampel
Keyword(s):  
Reference Gene ◽  
Reference Genes ◽  
Good Practice ◽  
Optimal Number ◽  
Suitable Reference Gene ◽  
Marine Bivalve ◽  
Mrna Levels ◽  
Experimental Conditions

AbstractReverse transcription real-time quantitative PCR (RT-qPCR) is the predominant method of choice for the quantification of mRNA transcripts of a selected gene of interest. Here reference genes are commonly used to normalize non-biological variation in mRNA levels and their appropriate selection is therefore essential for the accurate interpretation the collected data. In recent years the use of multiple validated references genes has been shown to substantially increase the robustness of the normalization. It is therefore considered good practice to experimentally validate putative reference genes under specific experimental conditions, determine the optimal number of reference genes to be employed, and report the method or methods used.Under this premise, we assessed the current state of reference gene base normalization in RT-qPCR bivalve ecotoxicology studies (post 2011), employing a systematic quantitative literature review. A total of 52 papers published met our criteria and were analysed for the gene or genes used, whether they employed multiple reference genes, as well as the validation method employed. In addition we performed a case study using primary hemocytes from the marine bivalve Ruditapes philippinarum after in vitro copper exposure. Herein we further critically discuss methods for reference gene validation, including the established algorithms geNorm, NormFinder and BestKeeper, as well as the popular online tool RefFinder.We identified that RT-qPCR normalization in bivalve ecotoxicology studies is largely performed using single reference genes, while less than 40% of the studies attempted to experimentally validate the expression stability of the reference genes used. 18s rRNA and β-Actin were the most popular genes, yet their un-validated use did introduce artefactual variance that altered the interpretation of the resulting data, while the use of appropriately validated reference genes did substantially improve normalization. Our findings further suggest that combining the results from multiple individual algorithms and calculating the overall best-ranked gene, as e.g. computed by the RefFinder tool, does not by default lead to the identification of the most suitable reference gene or combination of reference genes.

scholarly journals Identification of the most suitable reference gene for gene expression studies with development and abiotic stress response in Bromus sterilis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Madhab Kumar Sen ◽  
Kateřina Hamouzová ◽  
Pavlina Košnarová ◽  
Amit Roy ◽  
Josef Soukup
Keyword(s):  
Reference Gene ◽  
Herbicide Resistance ◽  
Reference Genes ◽  
Gene Selection ◽  
High Yield ◽  
Suitable Reference Gene ◽  
Grass Species ◽  
Experimental Conditions ◽  
Qrt Pcr ◽  
Suitable Reference

AbstractBromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can elucidate the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes was evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used software for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.

scholarly journals Glucan Biosynthesis Protein G Is a Suitable Reference Gene in Escherichia coli K-12

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Sean S. J. Heng ◽  
Oliver Y. W. Chan ◽  
Bryan M. H. Keng ◽  
Maurice H. T. Ling
Keyword(s):  
Escherichia Coli ◽  
Reference Gene ◽  
Reference Genes ◽  
Stability Index ◽  
Suitable Reference Gene ◽  
Protein G ◽  
Experimental Conditions ◽  
E Coli ◽  
Suitable Reference ◽  
K 12

The expressions of reference genes used in gene expression studies are assumed to be stable under most circumstances. However, a number of studies had demonstrated that such genes were found to vary under experimental conditions. In addition, genes that are stably expressed in an organ may not be stably expressed in other organs or other organisms, suggesting the need to identify reference genes for each organ and organism. This study aims at identifying stably expressed genes in Escherichia coli. Microarray datasets from E. coli substrain MG1655 and 1 dataset from W3110 were analysed. Coefficient of variance (COV) of was calculated and 10% of the lowest COV from 4631 genes common in the 3 MG1655 sets were analysed using NormFinder. Glucan biosynthesis protein G (mdoG), which is involved in cell wall synthesis, displayed the lowest weighted COV and weighted NormFinder Stability Index for the MG1655 datasets, while also showing to be the most stable in the dataset for substrain W3110, suggesting that mdoG is a suitable reference gene for E. coli K-12. Gene ontology over-representation analysis on the 39 genes suggested an over-representation of cell division, carbohydrate metabolism, and protein synthesis which supports the short generation time of E. coli.

scholarly journals Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages

2015 ◽  
Vol 2015 ◽  
pp. 1-14 ◽  
Author(s):  
Won-Jae Lee ◽  
Ryoung-Hoon Jeon ◽  
Si-Jung Jang ◽  
Ji-Sung Park ◽  
Seung-Chan Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Optimal Number ◽  
Experimental Conditions ◽  
Pearson’S Correlation ◽  
Qrt Pcr ◽  
Differentiated Cells ◽  
Pearson's Correlation ◽  
Suitable Reference

The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson’s correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson’s correlation revealed high correlation (r>0.9) between NF3and NFopt. Overall, the present study showed thatHMBS,YWHAZ,SDHA, andTBPare suitable reference genes for qRT-PCR in porcine MSCs.

scholarly journals Identification of the Most Suitable Reference Gene for Gene Expression Studies With Development and Abiotic Stress Response in Bromus Sterilis

2021 ◽  
Author(s):  
Madhab Kumar Sen ◽  
Katerina Hamouzova ◽  
Pavlina Kosnarova ◽  
Amit Roy ◽  
Josef Soukup
Keyword(s):  
Reference Gene ◽  
Herbicide Resistance ◽  
Reference Genes ◽  
Gene Selection ◽  
High Yield ◽  
Suitable Reference Gene ◽  
Grass Species ◽  
Experimental Conditions ◽  
Qrt Pcr ◽  
Suitable Reference

Abstract Bromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide-resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can contribute to elucidation of the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes were evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used softwares for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.

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 Current Practices for Reference Gene Selection in RT-qPCR of Aspergillus: Outlook and Recommendations for the Future

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 960
Author(s):  
Meagan Archer ◽  
Jianping Xu
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Selection ◽  
Quantitative Polymerase Chain Reaction ◽  
Specific Method ◽  
Experimental Conditions ◽  
Reference Gene Selection ◽  
Physiological Processes ◽  
The Stability

Aspergillus is a genus of filamentous fungi with vast geographic and ecological distributions. Species within this genus are clinically, agriculturally and biotechnologically relevant, leading to increasing interest in elucidating gene expression dynamics of key metabolic and physiological processes. Reverse-transcription quantitative Polymerase Chain Reaction (RT-qPCR) is a sensitive and specific method of quantifying gene expression. A crucial step for comparing RT-qPCR results between strains and experimental conditions is normalisation to experimentally validated reference gene(s). In this review, we provide a critical analysis of current reference gene selection and validation practices for RT-qPCR gene expression analyses of Aspergillus. Of 90 primary research articles obtained through our PubMed query, 17 experimentally validated the reference gene(s) used. Twenty reference genes were used across the 90 studies, with beta-tubulin being the most used reference gene, followed by actin, 18S rRNA and glyceraldehyde 3-phosphate dehydrogenase. Sixteen of the 90 studies used multiple reference genes for normalisation. Failing to experimentally validate the stability of reference genes can lead to conflicting results, as was the case for four studies. Overall, our review highlights the need to experimentally validate reference genes in RT-qPCR studies of Aspergillus.

scholarly journals Identification and selection of reference genes for gene expression analysis by quantitative real-time PCR in Suaeda glauca’s response to salinity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Meng Wang ◽  
Tingting Ren ◽  
Prince Marowa ◽  
Haina Du ◽  
Zongchang Xu
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Reference Genes ◽  
Suitable Reference Gene ◽  
Normalize Gene Expression ◽  
Suaeda Glauca ◽  
Suitable Reference ◽  
Germinating Seeds ◽  
Different Tissues

AbstractQuantitative real-time polymerase chain reaction (qPCR) using a stable reference gene is widely used for gene expression research. Suaeda glauca L. is a succulent halophyte and medicinal plant that is extensively used for phytoremediation and extraction of medicinal compounds. It thrives under high-salt conditions, which promote the accumulation of high-value secondary metabolites. However, a suitable reference gene has not been identified for gene expression standardization in S. glauca under saline conditions. Here, 10 candidate reference genes, ACT7, ACT11, CCD1, TUA5, UPL1, PP2A, DREB1D, V-H+-ATPase, MPK6, and PHT4;5, were selected from S. glauca transcriptome data. Five statistical algorithms (ΔCq, geNorm, NormFinder, BestKeeper, and RefFinder) were applied to determine the expression stabilities of these genes in 72 samples at different salt concentrations in different tissues. PP2A and TUA5 were the most stable reference genes in different tissues and salt treatments, whereas DREB1D was the least stable. The two reference genes were sufficient to normalize gene expression across all sample sets. The suitability of identified reference genes was validated with MYB and AP2 in germinating seeds of S. glauca exposed to different NaCl concentrations. Our study provides a foundational framework for standardizing qPCR analyses, enabling accurate gene expression profiling in S. glauca.

scholarly journals Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR in Macrobrachium Nipponense

2018 ◽  
Vol 19 (8) ◽  
pp. 2258 ◽  
Author(s):  
Yuning Hu ◽  
Hongtuo Fu ◽  
Hui Qiao ◽  
Shengming Sun ◽  
Wenyi Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Reference Gene ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Translation Initiation Factor ◽  
Suitable Reference Gene ◽  
Quantitative Real Time Pcr ◽  
Macrobrachium Nipponense ◽  
The Stability ◽  
Suitable Reference

Quantitative real-time PCR (qPCR) is widely used in molecular biology, although the accuracy of the quantitative results is determined by the stability of the reference genes used. Recent studies have investigated suitable reference genes for some crustaceans under various conditions, but studies in Macrobrachium nipponense are currently lacking. In this study, we selected the following seven genes from among 35 commonly used housekeeping genes as candidate qPCR reference genes for temporal and spatial expression: EIF (eukaryotic translation initiation factor 5A), 18S (18S ribosomal RNA), EF-1α (elongation factor-1α), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TUB (α-tubulin), β-act (β-actin), and RPL18 (Ribosomal protein L18). The stability of each reference gene was evaluated by GeNorm, NormFinder, BestKeeper, and comparative ∆C t methods, and was comprehensively ranked using RefFinder. RPL18 was shown to be the most suitable reference gene for adult M. nipponense tissues, while EIF was the most stable in different ovarian and embryo stages and in white spot syndrome virus infection, and β-act was the most stable reference gene under hypoxia stress. The reliability of the rankings was confirmed by RNA interference experiments. To the best of our knowledge, this represents the first systematic analysis of reference genes for qPCR experiments in M. nipponense, and the results will provide invaluable information for future research in closely related crustaceans.

scholarly journals Normalization of Gene Expression by Quantitative RT-PCR in Human Cell Line: comparison of 12 Endogenous Reference Genes

1970 ◽  
Vol 28 (6) ◽  
Author(s):  
Fowotade Adeola
Keyword(s):  
Real Time ◽  
Reference Genes ◽  
Human Cell Line ◽  
Optimal Number ◽  
Hek293 Cells ◽  
Mrna Levels ◽  
Expression Stability ◽  
Mrna Transcription ◽  
Reliable Quantification ◽  
Beta 2 Microglobulin

BACKGROUND: Polymerase Chain Reaction (PCR) has become an important diagnostic and research tool of modern molecular biology globally. Real-time PCR allows for rapid and reliable quantification of mRNA transcription. Reference genes are used as internal reaction control to normalise mRNA levels between different samples in order to allow for an exact comparison of mRNA transcription level.METHODS: In this study, twelve commonly used human reference genes were investigated in Human Embryonic Kidney Cell Lines (HEK293) using real-time qPCR with SYBR green. The genes included beta-2-microglobulin (B2M), glyceraldehyde-3- phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex subunit A (SDHA), and tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ). The stability of these reference genes was investigated using the geNorm application.RESULTS: The range of expression stability in the genes analysed was (from the most stable to the least stable): UBC, TOP1, ATP5B, CYC1, GAPDH, SDHA, YWHAZ, CTB, 18S, EIFA-2, B2M and RPL13A. The optimal number of reference targets in the experiment was calculated to be 2 (geNorm V<0.15) when comparing a normalization factor based on the 2 or 3 most stable targets).CONCLUSION: The expression stability varied greatly between the 12 candidate reference genes. UBC, TOP1, ATP5B, CYC1 and GAPDH respectively showed the highest stability in HEK293 cells based on both expression stability and expression level. Overall, our data suggest that UBC and TOP1show the least variation and the highest expression stability. This report validates the need for rational selection of reference genes for data normalization to ensure accuracy of quantitative PCR assays. 

scholarly journals Selection and validation of reference genes for quantitative gene expression analyses in persimmon (Diospyros kaki Thunb.) using real-time quantitative PCR

2019 ◽  
Vol 70 (4) ◽  
pp. 261-267
Author(s):  
Gaigai Du ◽  
Liyuan Wang ◽  
Huawei Li ◽  
Peng Sun ◽  
Jianmin Fu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Reference Genes ◽  
Developmental Stages ◽  
Suitable Reference Gene ◽  
Diospyros Kaki ◽  
Stable Gene ◽  
Expression Studies ◽  
Gene Expression Studies

Background and aims Persimmon (Diospyros kaki) is an economically important fruit tree species with complex flowering characteristics. To obtain accurate expression pattern analysis results, it is vital to select a reliable gene for the normalization of real-time quantitative polymerase chain reaction data. The aim of this study was to identify the optimal internal control gene among six candidate genes for gene expression analysis in different persimmon organs and developmental stages. Materials and methods This analysis was conducted using geNorm and NormFinder software to show differences in the stability of the six reference genes among tissues and floral developmental stages of the same plant. Results Although genes that exhibited moderate expression in NormFinder revealed slightly different expression stabilities than those obtained by geNorm, both sets of results showed that GAPDH was the best reference gene in different organs and floral buds at different developmental stages, whereas 18SrRNA was the least stable gene. Conclusions Based on the overall ranking, GAPDH is the most suitable reference gene and is highly recommended for gene expression studies in different organs and different developmental stages of persimmon. This study provides useful reference data for future gene expression studies and will contribute to improving the accuracy of gene expression results in persimmon.

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