scholarly journals Beyond initiation-limited translational bursting: the effects of burst size distributions on the stability of gene expression

2015 ◽  
Vol 7 (12) ◽  
pp. 1622-1632 ◽  
Author(s):  
Hiroyuki Kuwahara ◽  
Stefan T. Arold ◽  
Xin Gao
Keyword(s):  
Gene Expression ◽  
Burst Size ◽  
Size Distributions ◽  
The Stability

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.

scholarly journals Optimizations for identifying reference genes in bone and cartilage bioengineering

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Fei Xiong ◽  
Xiangyun Cheng ◽  
Chao Zhang ◽  
Roland Manfred Klar ◽  
Tao He
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Adipose Tissue ◽  
Real Time ◽  
Reference Gene ◽  
Muscle Tissue ◽  
Reference Genes ◽  
Target Genes ◽  
The Stability ◽  
And Cartilage

Abstract Background Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) remains one of the best-established techniques to assess gene expression patterns. However, appropriate reference gene(s) selection remains a critical and challenging subject in which inappropriate reference gene selction can distort results leading to false interpretations. To date, mixed opinions still exist in how to choose the most optimal reference gene sets in accodrance to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guideline. Therefore, the purpose of this study was to investigate which schemes were the most feasible for the identification of reference genes in a bone and cartilage bioengineering experimental setting. In this study, rat bone mesenchymal stem cells (rBMSCs), skeletal muscle tissue and adipose tissue were utilized, undergoing either chondrogenic or osteogenic induction, to investigate the optimal reference gene set identification scheme that would subsequently ensure stable and accurate interpretation of gene expression in bone and cartilage bioengineering. Results The stability and pairwise variance of eight candidate reference genes were analyzed using geNorm. The V0.15- vs. Vmin-based normalization scheme in rBMSCs had no significant effect on the eventual normalization of target genes. In terms of the muscle tissue, the results of the correlation of NF values between the V0.15 and Vmin schemes and the variance of target genes expression levels generated by these two schemes showed that different schemes do indeed have a significant effect on the eventual normalization of target genes. Three selection schemes were adopted in terms of the adipose tissue, including the three optimal reference genes (Opt3), V0.20 and Vmin schemes, and the analysis of NF values with eventual normalization of target genes showed that the different selection schemes also have a significant effect on the eventual normalization of target genes. Conclusions Based on these results, the proposed cut-off value of Vn/n + 1 under 0.15, according to the geNorm algorithm, should be considered with caution. For cell only experiments, at least rBMSCs, a Vn/n + 1 under 0.15 is sufficient in RT-qPCR studies. However, when using certain tissue types such as skeletal muscle and adipose tissue the minimum Vn/n + 1 should be used instead as this provides a far superior mode of generating accurate gene expression results. We thus recommended that when the stability and variation of a candidate reference genes in a specific study is unclear the minimum Vn/n + 1 should always be used as this ensures the best and most accurate gene expression value is achieved during RT-qPCR assays.

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 Viral-Mediated mRNA Degradation for Pathogenesis

Biomedicines ◽  
2018 ◽  
Vol 6 (4) ◽  
pp. 111
Author(s):  
Shujuan Du ◽  
Xiaoqing Liu ◽  
Qiliang Cai
Keyword(s):  
Gene Expression ◽  
Current Knowledge ◽  
Immune Surveillance ◽  
Viral Gene Expression ◽  
Mrna Degradation ◽  
Vital Role ◽  
Viral Gene ◽  
Antiviral Immune Response ◽  
Successful Infection ◽  
The Stability

Cellular RNA decay machinery plays a vital role in regulating gene expression by altering the stability of mRNAs in response to external stresses, including viral infection. In the primary infection, viruses often conquer the host cell’s antiviral immune response by controlling the inherently cellular mRNA degradation machinery to facilitate viral gene expression and establish a successful infection. This review summarizes the current knowledge about the diverse strategies of viral-mediated regulatory RNA shutoff for pathogenesis, and particularly sheds a light on the mechanisms that viruses evolve to elude immune surveillance during infection.

Identification of stable reference genes in differentiating human pluripotent stem cells

2015 ◽  
Vol 47 (6) ◽  
pp. 232-239 ◽  
Author(s):  
Gustav Holmgren ◽  
Nidal Ghosheh ◽  
Xianmin Zeng ◽  
Yalda Bogestål ◽  
Peter Sartipy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Gene Expression Data ◽  
Pluripotent Stem Cells ◽  
Reference Genes ◽  
Cell Types ◽  
Human Pluripotent Stem Cells ◽  
Expression Data ◽  
Large Variability ◽  
The Stability

Reference genes, often referred to as housekeeping genes (HKGs), are frequently used to normalize gene expression data based on the assumption that they are expressed at a constant level in the cells. However, several studies have shown that there may be a large variability in the gene expression levels of HKGs in various cell types. In a previous study, employing human embryonic stem cells (hESCs) subjected to spontaneous differentiation, we observed that the expression of commonly used HKG varied to a degree that rendered them inappropriate to use as reference genes under those experimental settings. Here we present a substantially extended study of the HKG signature in human pluripotent stem cells (hPSC), including nine global gene expression datasets from both hESC and human induced pluripotent stem cells, obtained during directed differentiation toward endoderm-, mesoderm-, and ectoderm derivatives. Sets of stably expressed genes were compiled, and a handful of genes (e.g., EID2, ZNF324B, CAPN10, and RABEP2) were identified as generally applicable reference genes in hPSCs across all cell lines and experimental conditions. The stability in gene expression profiles was confirmed by reverse transcription quantitative PCR analysis. Taken together, the current results suggest that differentiating hPSCs have a distinct HKG signature, which in some aspects is different from somatic cell types, and underscore the necessity to validate the stability of reference genes under the actual experimental setup used. In addition, the novel putative HKGs identified in this study can preferentially be used for normalization of gene expression data obtained from differentiating hPSCs.

scholarly journals Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Dorota M. Krzyżanowska ◽  
Anna Supernat ◽  
Tomasz Maciąg ◽  
Marta Matuszewska ◽  
Sylwia Jafra
Keyword(s):  
Gene Expression ◽  
Quorum Sensing ◽  
Reference Genes ◽  
Significance Threshold ◽  
Expression Studies ◽  
Reverse Transcription Quantitative Pcr ◽  
Gene Expression Studies ◽  
The Stability ◽  
Selection Of

Abstract Reverse transcription quantitative PCR (RT-qPCR), a method of choice for quantification of gene expression changes, requires stably expressed reference genes for normalization of data. So far, no reference genes were established for the Alphaproteobacteria of the genus Ochrobactrum. Here, we determined reference genes for gene expression studies in O. quorumnocens A44. Strain A44 was cultured under 10 different conditions and the stability of expression of 11 candidate genes was evaluated using geNorm, NormFinder and BestKeeper. Most stably expressed genes were found to be rho, gyrB and rpoD. Our results can facilitate the choice of reference genes in the related Ochrobactrum strains. O. quorumnocens A44 is able to inactivate a broad spectrum of N-acyl homoserine lactones (AHLs) – the quorum sensing molecules of many Gram-negative bacteria. This activity is attributed to AiiO hydrolase, yet it remains unclear whether AHLs are the primary substrate of this enzyme. Using the established RT-qPCR setup, we found that the expression of the aiiO gene upon exposure to two AHLs, C6-HLS and 3OC12-HSL, does not change above the 1-fold significance threshold. The implications of this finding are discussed in the light of the role of quorum sensing-interfering enzymes in the host strains.

scholarly journals Probability in transcriptional regulation and its implications for leukocyte differentiation and inducible gene expression

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2323-2328 ◽  
Author(s):  
David A. Hume
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Mammalian Cells ◽  
Cell Lineage ◽  
Monoallelic Expression ◽  
Stem Cell Lineage ◽  
The Stability ◽  
Stochastic Phenomena ◽  
Inducible Genes ◽  
Probabilistic Nature

The phenotype of individual hematopoietic cells, like all other differentiated mammalian cells, is determined by selective transcription of a subset of the genes encoded within the genome. This overview summarizes the recent evidence that transcriptional regulation at the level of individual cells is best described in terms of the regulation of the probability of transcription rather than the rate. In this model, heterogeneous gene expression among populations of cells arises by chance, and the degree of heterogeneity is a function of the stability of the mRNA and protein products of individual genes. The probabilistic nature of transcriptional regulation provides one explanation for stochastic phenomena, such as stem cell lineage commitment, and monoallelic expression of inducible genes, such as lymphokines and cytokines.

scholarly journals Tissue-specific evaluation of suitable reference genes for RT-qPCR in the pond snail, Lymnaea stagnalis

2019 ◽  
Author(s):  
Alexander P Young ◽  
Carmen F Landry ◽  
Daniel J Jackson ◽  
Russell C Wyeth
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Lymnaea Stagnalis ◽  
Elongation Factor ◽  
Pond Snail ◽  
Tissue Samples ◽  
Systematic Assessment ◽  
Wide Range ◽  
The Stability ◽  
Suitable Reference

Reverse transcription quantitative PCR (RT-qPCR) is a robust technique for the quantification and comparison of gene expression across multiple tissues. To obtain reliable results, one or more reference genes must be employed to normalize expression measurements among treatments or tissue samples. Candidate reference genes must be validated to ensure that they are stable prior to use in qPCR experiments. The pond snail (Lymnaea stagnalis) is a common research organism, particularly in the areas of learning and memory, and is an emerging target for qPCR experimentation. However, no systematic assessment of reference genes has been performed in this animal. Therefore, the aim of our research was to identify stable reference genes to normalize gene expression data from a variety of tissues in L. stagnalis. We evaluated a panel of seven reference genes across six different tissues in L. stagnalis with RT-qPCR. The genes included: elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (ACTB), beta-tubulin (TUBB), ubiquitin (UBI), prenylated rab acceptor protein 1 (Rapac1), and a voltage gated potassium channel (VGKC). These genes exhibited a wide range of expression levels among tissues. The stability of each of the genes was consistent when measured by any of the standard stability assessment algorithms: geNorm, NormFinder, BestKeeper and RefFinder. Our data indicate that GAPDH and EF1α are highly stable in the tissues that we examined (central nervous system, tentacles, lips, penis, foot, mantle) as well as in pooled analyses. We do not recommend VGKC for use in RT-qPCR experiments due to its relatively low expression stability. Our results were generally congruent with those obtained from similar studies in other molluscs. Given that a minimum of two reference genes are recommended for data normalization, we suggest GAPDH and EF1α are a strong option for multi-tissue analyses of RT-qPCR data in Lymnaea stagnalis.

scholarly journals The effect of hairpin loop on the structure and gene expression activity of the long-loop G-quadruplex

2021 ◽  
Author(s):  
Subramaniyam Ravichandran ◽  
Maria Razzaq ◽  
Nazia Parveen ◽  
Ambarnil Ghosh ◽  
Kyeong Kyu Kim
Keyword(s):  
Gene Expression ◽  
Rna Structure ◽  
Biological Significance ◽  
Cellular Functions ◽  
Hairpin Loops ◽  
G Quadruplex ◽  
Reporter Assays ◽  
Expression Activity ◽  
The Stability ◽  
And Function

Abstract G-quadruplex (G4), a four-stranded DNA or RNA structure containing stacks of guanine tetrads, plays regulatory roles in many cellular functions. So far, conventional G4s containing loops of 1–7 nucleotides have been widely studied. Increasing experimental evidence suggests that unconventional G4s, such as G4s containing long loops (long-loop G4s), play a regulatory role in the genome by forming a stable structure. Other secondary structures such as hairpins in the loop might thus contribute to the stability of long-loop G4s. Therefore, investigation of the effect of the hairpin-loops on the structure and function of G4s is required. In this study, we performed a systematic biochemical investigation of model G4s containing long loops with various sizes and structures. We found that the long-loop G4s are less stable than conventional G4s, but their stability increased when the loop forms a hairpin (hairpin-G4). We also verified the biological significance of hairpin-G4s by showing that hairpin-G4s present in the genome also form stable G4s and regulate gene expression as confirmed by in cellulo reporter assays. This study contributes to expanding the scope and diversity of G4s, thus facilitating future studies on the role of G4s in the human genome.

scholarly journals Validation of Internal Control Genes for Quantitative Real-Time PCR Gene Expression Analysis in Morchella

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2331 ◽  
Author(s):  
Qianqian Zhang ◽  
Wei Liu ◽  
Yingli Cai ◽  
A-Feng Lan ◽  
Yinbing Bian
Keyword(s):  
Gene Expression ◽  
Candidate Genes ◽  
Expression Analysis ◽  
Internal Control ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Stable Gene ◽  
Experimental Conditions ◽  
The Stability ◽  
Vacuolar Protein

The reliability of qRT-PCR results depend on the stability of reference genes used for normalization, suggesting the necessity of identification of reference genes before gene expression analysis. Morels are edible mushrooms well-known across the world and highly prized by many culinary kitchens. Here, several candidate genes were selected and designed according to the Morchella importuna transcriptome data. The stability of the candidate genes was evaluated with geNorm and NormFinder under three different experimental conditions, and several genes with excellent stability were selected. The extensive adaptability of the selected genes was tested in ten Morchella species. Results from the three experimental conditions revealed that ACT1 and INTF7 were the most prominent genes in Morchella, CYC3 was the most stable gene in different development stages, INTF4/AEF3 were the top-ranked genes across carbon sources, while INTF3/CYC3 pair showed the robust stability for temperature stress treatment. We suggest using ACT1, AEF3, CYC3, INTF3, INTF4 and INTF7 as reference genes for gene expression analysis studies for any of the 10 Morchella strains tested in this study. The stability and practicality of the gene, vacuolar protein sorting (INTF3), vacuolar ATP synthase (INTF4) and14-3-3 protein (INTF7) involving the basic biological processes were validated for the first time as the candidate reference genes for quantitative PCR. Furthermore, the stability of the reference genes was found to vary under the three different experimental conditions, indicating the importance of identifying specific reference genes for particular conditions.

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