scholarly journals Selection and Evaluation of Reference Genes for ddPCR-Based Transcript Abundance Studies in Oidiodendron Maius Across Varying Carbon Sources

2021 ◽  
Author(s):  
Erin Feldman ◽  
Elena Martino ◽  
Annegret Kohler ◽  
Daniel Durall ◽  
Melanie Jones
Keyword(s):  
Stability Analysis ◽  
Differentially Expressed Genes ◽  
Reference Genes ◽  
Geometric Mean ◽  
Mycorrhizal Fungus ◽  
Carbon Sources ◽  
Transcript Abundance ◽  
Differentially Expressed ◽  
Transcriptional Responses ◽  
Oidiodendron Maius

Abstract Background When identifying transcript abundance in response to treatment, accurate quantification is critical, especially when examining subtle differences in expression. In particular, data normalization is necessary to account for differences among samples including those associated with RNA quantity and quality. Due to the capacity of droplet digital PCR to absolutely quantify the copy number of the target gene in a given sample, normalization, such as the use of an internal control gene, has not customarily been considered obligatory. Decades of quantitative PCR research have shown, however, that the use of endogenous controls undoubtedly aid in correcting sample variability. With our limited knowledge of gene function in many fungi, typical ‘housekeeping genes’ commonly used as internal references may not be relevant in these organisms. This study aimed to identify and validate suitable reference genes for transcript abundance studies in Oidiodendron maius, a globally distributed, model ericoid mycorrhizal fungus. Results A shortlist of 251 non-differentially expressed genes was generated from RNA-Seq analyses of O. maius grown on three different carbon sources or in symbiosis with Vaccinium myrtillus. Subsequently, a set of criteria (stable expression, valid annotation and relatively high expression) was applied to select three candidate reference genes. These three genes were validated across a further eleven carbon sources using ddPCR and the application of geNorm and NormFinder stability analysis algorithms. Expression stability analysis of three genes - EfTu, vma, and sar - confirmed their reliability as internal references; the geometric mean of their expression values demonstrated the highest stability as a normalization factor.Conclusions We propose the use of the geometric mean of O. maius genes EfTu, vma and sar as a reference tool to normalize RNA expression in ddPCR assays. These newly selected and validated reference genes will increase reliability and reproducibility when studying transcriptional responses of O. maius at different developmental stages and/or under a range of physiological conditions. In addition, the list of 251 non-differentially expressed genes can serve as a valuable resource for selecting reference genes for related experiments and enhances the limited information available on O. maius.

Long-Living Budding Yeast Cell Subpopulation Induced by Ethanol/Acetate and Respiration

2019 ◽  
Vol 75 (8) ◽  
pp. 1448-1456 ◽  
Author(s):  
Young-Yon Kwon ◽  
Seung-Soo Kim ◽  
Han-Jun Lee ◽  
Seo-Hyeong Sheen ◽  
Kyoung Heon Kim ◽  
...  
Keyword(s):  
Differentially Expressed Genes ◽  
Budding Yeast ◽  
Gradient Centrifugation ◽  
Carbon Sources ◽  
Cell Types ◽  
Glucose Sensing ◽  
Differentially Expressed ◽  
Cell Subpopulation ◽  
Genes Encoding ◽  
Cell Transcriptome

Abstract Budding yeast generate heterogeneous cells that can be separated into two distinctive cell types: short-living low-density and long-living high-density (HD) cells by density gradient centrifugation. We found that ethanol and acetate induce formation of HD cells, and mitochondrial respiration is required. From their transcriptomes and metabolomes, we found upregulated differentially expressed genes in HD cells involved in the RGT2/RGT1 glucose sensing pathway and its downstream genes encoding hexose transporters. For HD cells, we determined an abundance of various carbon sources including glucose, lactate, pyruvate, trehalose, mannitol, mannose, and galactose. Other upregulated differentially expressed genes in HD cells were involved in the TORC1–SCH9 signaling pathway and its downstream genes involved in cytoplasmic translation. We also measured an abundance of free amino acids in HD cells including valine, proline, isoleucine, and glutamine. These characteristics of the HD cell transcriptome and metabolome may be important conditions for maintaining a long-living phenotype.

scholarly journals Identification and characterization of differentially expressed genes in Caenorhabditis elegans in response to pathogenic and nonpathogenic Stenotrophomonas maltophilia

2019 ◽  
Author(s):  
Leah J Radeke ◽  
Michael Herman
Keyword(s):  
Candidate Genes ◽  
Differentially Expressed Genes ◽  
Stenotrophomonas Maltophilia ◽  
Defense Mechanisms ◽  
Virulent Strain ◽  
Differentially Expressed ◽  
Transcriptional Responses ◽  
Transcriptomic Response ◽  
C Elegans ◽  
Realistic Interaction

Abstract Background: Stenotrophomonas maltophilia is an emerging nosocomial pathogen that causes infection in immunocompromised patients. S. maltophilia isolates are genetically diverse, contain diverse virulence factors, and are variably pathogenic within several host species. Members of the Stenotrophomonas genus are part of the native microbiome of C. elegans , being found in greater relative abundance within the worm than its environment, suggesting that these bacteria accumulate within C. elegans . Thus, study of the C. elegans-Stenotrophomonas interaction is of both medical and ecological significance. To identify host defense mechanisms, we analyzed the C. elegans transcriptomic response to S. maltophilia strains of varying pathogenicity: K279a, an avirulent clinical isolate, JCMS, a virulent strain isolated in association with soil nematodes near Manhattan, KS, and JV3, an even more virulent environmental isolate. Results: Overall, we found 145 genes that are commonly differentially expressed in response to pathogenic S. maltophilia strains, 89% of which are upregulated, with many even further upregulated in response to JV3 as compared to JCMS. There are many more JV3-specific differentially expressed genes (225, 11% upregulated) than JCMS-specific differentially expressed genes (14, 86% upregulated), suggesting JV3 has unique pathogenic mechanisms that could explain its increased virulence. We used connectivity within a gene network model to choose pathogen-specific and strain-specific differentially expressed candidate genes for functional analysis. Mutations in 13 of 22 candidate genes caused significant differences in C. elegans survival in response to at least one S. maltophilia strain, although not always the strain that induced differential expression, suggesting a dynamic response to varying levels of pathogenicity. Conclusions: Variation in observed pathogenicity and differences in host transcriptional responses to S. maltophilia strains reveal that strain-specific mechanisms play important roles in S. maltophilia pathogenesis. Furthermore, utilizing bacteria closely related to strains found in C. elegans natural environment provides a more realistic interaction for understanding host-pathogen response.

scholarly journals Palmitate and Fructose Interact to Induce Human Hepatocytes to Produce Pro-Fibrotic Transcriptional Responses in Hepatic Stellate Cells Exposed to Conditioned Media

2020 ◽  
Vol 54 (5) ◽  
pp. 1068-1082
Keyword(s):  
Differential Expression ◽  
Differentially Expressed Genes ◽  
Hepatic Stellate Cells ◽  
Stellate Cell ◽  
Stellate Cells ◽  
Human Hepatocytes ◽  
Conditioned Media ◽  
Differentially Expressed ◽  
Transcriptional Responses ◽  
Significant Differential Expression

BACKGROUND/AIMS: Excessive consumption of dietary fat and sugar is associated with an elevated risk of nonalcoholic fatty liver disease (NAFLD). Hepatocytes exposed to saturated fat or sugar exert effects on nearby hepatic stellate cells (HSCs); however, the mechanisms by which this occurs are poorly understood. We sought to determine whether paracrine effects of hepatocytes exposed to palmitate and fructose produced profibrotic transcriptional responses in HSCs. METHODS: We performed expression profiling of mRNA and lncRNA from HSCs treated with conditioned media (CM) from human hepatocytes treated with palmitate (P), fructose (F), or both (PF). RESULTS: In HSCs exposed to CM from palmitate-treated hepatocytes, we identified 374 mRNAs and 607 lncRNAs showing significant differential expression (log2 foldchange ≥ |1|; FDR ≤0.05) compared to control cells. In HSCs exposed to CM from PF-treated hepatocytes, the number of differentially expressed genes was much higher (1198 mRNAs and 3348 lncRNAs); however, CM from fructose-treated hepatocytes elicited no significant changes in gene expression. Pathway analysis of differentially expressed genes showed enrichment for hepatic fibrosis and hepatic stellate cell activation in P- (FDR =1.30E-04) and PF-(FDR =9.24E-06)
groups. We observed 71 lncRNA/nearby mRNA pairs showing differential expression under PF conditions. There were 90 mRNAs and 264 lncRNAs strongly correlated between the PF group and differentially expressed transcripts from a comparison of activated and quiescent HSCs, suggesting that some of the transcriptomic changes occurring in response to PF overlap with HSC activation. CONCLUSION: The results reported here have implications for dietary modifications in the prevention and treatment of NAFLD.

scholarly journals A red herring in zebrafish genetics: allele-specific gene expression can underlie altered transcript abundance in zebrafish mutants

2021 ◽  
Author(s):  
Richard J White ◽  
Eirinn Mackay ◽  
Stephen W Wilson ◽  
Elisabeth M Busch-Nentwich
Keyword(s):  
Differentially Expressed Genes ◽  
Differentially Expressed Gene ◽  
Transcript Abundance ◽  
Differentially Expressed ◽  
Model Organisms ◽  
Specific Gene ◽  
Wild Type ◽  
Specific Expression ◽  
Genomic Location ◽  
Allele Specific

In model organisms, RNA sequencing is frequently used to assess the effect of genetic mutations on cellular and developmental processes. Typically, animals heterozygous for a mutation are crossed to produce offspring with different genotypes. Resultant embryos are grouped by genotype to compare homozygous mutant embryos to heterozygous and wild-type siblings. Genes that are differentially expressed between the groups are assumed to reveal insights into the pathways affected by the mutation. Here we show that in zebrafish, differentially expressed genes are often overrepresented on the same chromosome as the mutation due to different levels of expression of alleles from different genetic backgrounds. Using an incross of haplotype-resolved wild-type fish, we found evidence of widespread allele-specific expression, which appears as differential expression when comparing embryos homozygous for a region of the genome to their siblings. When analysing mutant transcriptomes, this means that differentially expressed genes on the same chromosome as a mutation of interest may not be caused by that mutation. Typically, the genomic location of a differentially expressed gene is not considered when interpreting its importance with respect to the phenotype. This could lead to pathways being erroneously implicated or overlooked due to the noise of spurious differentially expressed genes on the same chromosome as the mutation. These observations have implications for the interpretation of RNA-seq experiments involving outbred animals and non-inbred model organisms.

scholarly journals Regulatory Crosstalk of Doxorubicin, Estradiol and TNFα Combined Treatment in Breast Cancer-derived Cell Lines

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Isar Nassiri ◽  
Alberto Inga ◽  
Erna Marija Meškytė ◽  
Federica Alessandrini ◽  
Yari Ciribilli ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
Differentially Expressed Genes ◽  
Combination Treatment ◽  
Combined Treatment ◽  
Enrichment Analysis ◽  
Differentially Expressed ◽  
Transcriptional Responses ◽  
Predisposing Factor ◽  
Inflammatory Microenvironment

Abstract We present a new model of ESR1 network regulation based on analysis of Doxorubicin, Estradiol, and TNFα combination treatment in MCF-7. We used Doxorubicin as a therapeutic agent, TNFα as marker and mediator of an inflammatory microenvironment and 17β-Estradiol (E2) as an agonist of Estrogen Receptors, known predisposing factor for hormone-driven breast cancer, whose pharmacological inhibition reduces the risk of breast cancer recurrence. Based on the results of transcriptomics analysis, we found 71 differentially expressed genes that are specific for the combination treatment with Doxorubicin + Estradiol + TNFα in comparison with single or double treatments. The responsiveness to the triple treatment was examined for seven genes by qPCR, of which six were validated, and then extended to four additional cell lines differing for p53 and/or ER status. The results of differential regulation enrichment analysis highlight the role of the ESR1 network that included 36 of 71 specific differentially expressed genes. We propose that the combined activation of p53 and NF-kB transcription factors significantly influences ligand-dependent, ER-driven transcriptional responses, also of the ESR1 gene itself. These results provide a model of coordinated interaction of TFs to explain the Doxorubicin, E2 and TNFα induced repression mechanisms.

scholarly journals The synergistic interaction of thermal stress coupled with overstocking strongly modulates the transcriptomic activity and immune capacity of rainbow trout (Oncorhynchus mykiss)

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Alexander Rebl ◽  
Tomáš Korytář ◽  
Andreas Borchel ◽  
Ralf Bochert ◽  
Joanna Ewa Strzelczyk ◽  
...  
Keyword(s):  
Rainbow Trout ◽  
High Temperature ◽  
Critical Temperature ◽  
Differentially Expressed Genes ◽  
Reference Group ◽  
Synergistic Effects ◽  
Stocking Density ◽  
Transcript Abundance ◽  
Condition Index ◽  
Differentially Expressed

Abstract The objective of the present study is to identify and evaluate informative indicators for the welfare of rainbow trout exposed to (A) a water temperature of 27 °C and (B) a stocking density of 100 kg/m3 combined with a temperature of 27 °C. The spleen-somatic and condition index, haematocrit and the concentrations of haemoglobin, plasma cortisol and glucose revealed non-significant differences between the two stress groups and the reference group 8 days after the onset of the experiments. The transcript abundance of almost 1,500 genes was modulated at least twofold in in the spleen of rainbow trout exposed to a critical temperature alone or a critical temperature combined with crowding as compared to the reference fish. The number of differentially expressed genes was four times higher in trout that were simultaneously challenged with high temperature and crowding, compared to trout challenged with high temperature alone. Based on these sets of differentially expressed genes, we identified unique and common tissue- and stress type-specific pathways. Furthermore, our subsequent immunologic analyses revealed reduced bactericidal and inflammatory activity and a significantly altered blood-cell composition in challenged versus non-challenged rainbow trout. Altogether, our data demonstrate that heat and overstocking exert synergistic effects on the rainbow trout’s physiology, especially on the immune system.

A novel method to prioritize RNAseq data for post-hoc analysis based on absolute changes in transcript abundance

2015 ◽  
Vol 14 (3) ◽  
Author(s):  
Patrick McNutt ◽  
Ian Gut ◽  
Kyle Hubbard ◽  
Phil Beske
Keyword(s):  
Differentially Expressed Genes ◽  
Transcript Abundance ◽  
Fold Change ◽  
Differentially Expressed ◽  
Post Hoc Analysis ◽  
Rnaseq Data ◽  
Novel Method ◽  
Post Hoc

AbstractThe use of fold-change (FC) to prioritize differentially expressed genes (DEGs) for

scholarly journals Multi-organ transcriptomic landscape of Ambystoma velasci metamorphosis

2020 ◽  
Author(s):  
Palacios-Martínez Janet ◽  
Caballero-Pérez Juan ◽  
Espinal-Centeno Annie ◽  
Marquez-Chavoya Gilberto ◽  
Lomelí Hilda ◽  
...  
Keyword(s):  
Differentially Expressed Genes ◽  
Developmental Process ◽  
Global Analysis ◽  
Expression Patterns ◽  
Coronary Vessels ◽  
Aquatic Organisms ◽  
Gene Clusters ◽  
Differentially Expressed ◽  
Transcriptional Responses ◽  
Terrestrial Environment

Metamorphosis is a postembryonic developmental process that involves morphophysiological and behavioral changes, allowing organisms to adapt into a novel environment. In some amphibians, aquatic organisms undergo metamorphosis to adapt in a terrestrial environment. These organisms experience major changes in their circulatory, respiratory, digestive, excretory and reproductive systems. We performed a transcriptional global analysis of heart, lung and gills during diverse stages of Ambystoma velasci metamorphosis. In our analyses, we identified eight gene clusters for each organ, according to the expression patterns of differentially expressed genes. We found 4,064 differentially expressed genes in the heart, 4,107 in the lung and 8,265 in the gills. Among the differentially expressed genes in the heart, we observed genes involved in the differentiation of cardiomyocytes in the interatrial zone, vasculogenesis and in the maturation of coronary vessels. In the lung, we found genes differentially expressed related to angiogenesis, alveolarization and synthesis of the surfactant protein. In the case of the gills, the most prominent biological processes identified are degradation of extracellular matrix, apoptosis and keratin production. Our study sheds light on the transcriptional responses and the pathways involved in the transformation of the facultative metamorphic salamander A. velasci in an organ-specific manner.

scholarly journals Performance of gene expression analyses using de novo assembled transcripts in polyploid species

2018 ◽  
Author(s):  
Ling-Yun Chen ◽  
Diego F. Morales-Briones ◽  
Courtney N. Passow ◽  
Ya Yang
Keyword(s):  
Gene Expression ◽  
Differentially Expressed Genes ◽  
Reference Genes ◽  
De Novo ◽  
Differentially Expressed ◽  
Supplementary Information ◽  
Network Analyses ◽  
Gene Expression Analyses ◽  
Polyploidy Event ◽  
Differential Gene

AbstractMotivationQuality of gene expression analyses using de novo assembled transcripts in species experienced recent polyploidization is yet unexplored.ResultsFive plant species with various polyploidy history were used for differential gene expression (DGE) analyses. DGE analyses using putative genes inferred by Trinity performed similar to or better than Corset and Grouper in precision, but lower in sensitivity. In species that lack polyploidy event in the past few million years, DGE analyses using de novo assembled transcriptome identified 50–76% of the differentially expressed genes recovered by mapping reads to the reference genes. However, in species with more recent polyploidy event, the percentage decreased to 7–30%. In addition, 7–89% of differentially expressed genes from de novo assembly are contaminations. Gene co-expression network analyses using de novo assemblies vs. mapping to the reference genes recovered the same module that significantly correlated with treatment in one of the five species tested.Availability and ImplementationCommands and scripts used in this study are available at https://bitbucket.org/lychen83/chen_et_al_2018_benchmark_dge/; Analysis files are available at Dryad doi: [email protected] informationSupplementary data are available at Bioinformatics online

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