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 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 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 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 Screening of reference genes in real-time PCR for Radopholus similis

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6253 ◽  
Author(s):  
Jun-Yi Li ◽  
Wan-Zhu Chen ◽  
Si-Hua Yang ◽  
Chun-Ling Xu ◽  
Xin Huang ◽  
...  
Keyword(s):  
Real Time ◽  
Reference Gene ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Developmental Stages ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Suitable Reference Gene ◽  
Radopholus Similis ◽  
Suitable Reference

Six candidate reference genes were chosen from the transcriptome database of Radopholus similis using the bioinformatics method, including four conventional reference genes (actin, Eukaryotic translation initiation factor 5A (eIF5A), Tubulin alpha (a-tubulin), ubiquitin (UBI)) and two new candidate reference genes (Ribosomal protein S21 (Rps21) and Serine/threonine protein phosphatase PP1-β catalytic subunit (β-PP1)). In addition, a traditional reference gene 18S ribosomal RNA (18S rRNA) obtained from NCBI databases was also added to the analysis. Real-time PCR was used to detect the expression of seven candidate reference genes in six populations of R. similis and four developmental stages (female, male, larva and egg) of a population. The stability of the expression of candidate genes was evaluated by three software programs, BestKeeper, geNorm and NormFinder. The results showed that eIF5A is the most suitable reference gene for gene functional research of different populations, while both Rps21 and eIF5A are the most suitable reference genes for different developmental stages of a population. Therefore, eIF5A is the best reference gene for studying R. similis. However, one defect of this study is that only seven candidate reference genes were analyzed; ideally, more genes should be tested.

scholarly journals Selection and Stability Validation of Reference Gene Candidates for Transcriptional Analysis in Rousettus Aegyptiacus

2021 ◽  
Author(s):  
Virginia Friedrichs ◽  
Anne Balkema-Buschmann ◽  
Anca Dorhoi ◽  
Gang Pei
Keyword(s):  
Body Temperature ◽  
Reference Gene ◽  
Reference Genes ◽  
Core Body Temperature ◽  
Transcriptional Analysis ◽  
Suitable Reference Gene ◽  
Type I ◽  
Expression Stability ◽  
Qrt Pcr ◽  
Suitable Reference

Abstract Bats are the only mammals capable of powered flight and their body temperature can reach up to 42°C during flight. Additionally, bats display robust type I IFN interferon (IFN-I) responses and some species constitutively express IFN-α. Reference genes with stable expression under temperature oscillations and IFN-I release are therefore critical for normalization of quantitative reverse-transcription polymerase chain reaction (qRT-PCR) data in bats. The expression stability of reference genes in Rousettus aegyptiacus remains elusive, although this species is frequently used in the infection research. We selected ACTB, EEF1A1, GAPDH and PGK1 as candidate reference genes and evaluated their expression stability in various tissues and cells from this model bat species upon IFN-I treatment at 37°C and 40°C by qRT-PCR. We employed two statistical algorithms, BestKeeper and NormFinder, and found that EEF1A1 exhibited the highest stability under all tested conditions. ACTB and GAPDH displayed unstable expression at 40°C and upon IFN-I treatment, respectively. By normalizing to EEF1A1, we uncovered that GAPDH expression was significantly induced by IFN‑I in R. aegyptiacus. Our study identifies EEF1A1 as the most suitable reference gene for qRT-PCR studies and unveils the induction of GAPDH expression by IFN-I in R. aegyptiacus. These findings are pertinent to other bat species and even bear relevance for non-volant mammals that show physiological fluctuations of core body temperature.

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 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 Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Zhaoping Yan ◽  
Jinhang Gao ◽  
Xiuhe Lv ◽  
Wenjuan Yang ◽  
Shilei Wen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Pancreatitis ◽  
Reference Gene ◽  
Reference Genes ◽  
Suitable Reference Gene ◽  
Expression Stability ◽  
Rt Pcr ◽  
Comprehensive Method ◽  
Suitable Combination ◽  
Suitable Reference

The analysis of differences in gene expression is dependent on normalization using reference genes. However, the expression of many of these reference genes, as evaluated by quantitative RT-PCR, is upregulated in acute pancreatitis, so they cannot be used as the standard for gene expression in this condition. For this reason, we sought to identify a stable reference gene, or a suitable combination, for expression analysis in acute pancreatitis. The expression stability of 10 reference genes (ACTB, GAPDH, 18sRNA, TUBB, B2M, HPRT1, UBC, YWHAZ, EF-1α, and RPL-13A) was analyzed using geNorm, NormFinder, and BestKeeper software and evaluated according to variations in the raw Ct values. These reference genes were evaluated using a comprehensive method, which ranked the expression stability of these genes as follows (from most stable to least stable): RPL-13A, YWHAZ > HPRT1 > GAPDH > UBC > EF-1α> 18sRNA > B2M > TUBB > ACTB. RPL-13A was the most suitable reference gene, and the combination of RPL-13A and YWHAZ was the most stable group of reference genes in our experiments. The expression levels of ACTB, TUBB, and B2M were found to be significantly upregulated during acute pancreatitis, whereas the expression level of 18sRNA was downregulated. Thus, we recommend the use of RPL-13A or a combination of RPL-13A and YWHAZ for normalization in qRT-PCR analyses of gene expression in mouse models of acute pancreatitis.

scholarly journals Identification and validation of potential reference gene for effective dsRNA knockdown analysis in Chilo partellus

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Olawale Samuel Adeyinka ◽  
Bushra Tabassum ◽  
Idrees Ahmad Nasir ◽  
Iqra Yousaf ◽  
Imtiaz Ahmad Sajid ◽  
...  
Keyword(s):  
Reference Gene ◽  
Chilo Partellus ◽  
Reference Genes ◽  
Gene Selection ◽  
Developmental Stages ◽  
Stable Reference Gene ◽  
Suitable Reference Gene ◽  
Potential Reference Gene ◽  
Alternate Control ◽  
Suitable Reference

Abstract Chilo partellus is an invasive polyphagous pest that has not been effectively managed with chemical pesticides. To select potential dsRNAs for use in an alternate control strategy, it is crucial to identify and evaluate stable reference genes for knockdown expression studies. This study evaluates the expression stability of seven candidate reference genes in C. partellus larvae fed on crude bacterially-expressed dsRNAs and purified dsRNAs at different time intervals, as well as the developmental stages and sexes. The expression stabilities of the reference genes were evaluated with different software programmes, such as BestKeeper, NormFinder, deltaCt, geNorm, and RefFinder. The overall results rank ELF as the most stably expressed reference gene when larvae were fed with crude bacteria-induced dsRNAs and purified dsRNA. However, Tubulin and HSP70 were more stable under different developmental stages and sexes. The expression levels of larvae that were fed crude bacteria-induced dsRNAs of Chitinase and Acetylcholinesterase were normalized with the four most stable reference genes (ELF, HSP70, V-ATPase and Tubulin) and the least stable reference gene (18S and HSP70) based on the geNorm algorithm. The least stable reference gene showed inconsistent knockdown expression, thereby confirming that the validation of a suitable reference gene is crucial to improve assay accuracy for dsRNA-targeted gene selection in C. partellus.

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