scholarly journals Transcriptional profiling at whole population and single cell levels reveals somatosensory neuron molecular diversity

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Isaac M Chiu ◽  
Lee B Barrett ◽  
Erika K Williams ◽  
David E Strochlic ◽  
Seungkyu Lee ◽  
...  
Keyword(s):  
Clustering Analysis ◽  
Molecular Diversity ◽  
Transcriptional Profiling ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Molecular Signatures ◽  
Qrt Pcr ◽  
Somatosensory Neurons ◽  
Overlapping Classes ◽  
Somatosensory Nervous System

The somatosensory nervous system is critical for the organism's ability to respond to mechanical, thermal, and nociceptive stimuli. Somatosensory neurons are functionally and anatomically diverse but their molecular profiles are not well-defined. Here, we used transcriptional profiling to analyze the detailed molecular signatures of dorsal root ganglion (DRG) sensory neurons. We used two mouse reporter lines and surface IB4 labeling to purify three major non-overlapping classes of neurons: 1) IB4+SNS-Cre/TdTomato+, 2) IB4−SNS-Cre/TdTomato+, and 3) Parv-Cre/TdTomato+ cells, encompassing the majority of nociceptive, pruriceptive, and proprioceptive neurons. These neurons displayed distinct expression patterns of ion channels, transcription factors, and GPCRs. Highly parallel qRT-PCR analysis of 334 single neurons selected by membership of the three populations demonstrated further diversity, with unbiased clustering analysis identifying six distinct subgroups. These data significantly increase our knowledge of the molecular identities of known DRG populations and uncover potentially novel subsets, revealing the complexity and diversity of those neurons underlying somatosensation.

scholarly journals Commitment of Autologous Human Multipotent Stem Cells on Biomimetic Poly-L-Lactic Acid-Based Scaffolds Is Strongly Influenced by Structure and Concentration of Carbon Nanomaterial

Nanomaterials ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 415
Author(s):  
Marika Tonellato ◽  
Monica Piccione ◽  
Matteo Gasparotto ◽  
Pietro Bellet ◽  
Lucia Tibaudo ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Carbon Nanomaterials ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Carbon Nanomaterial ◽  
Multipotent Stem Cells ◽  
Cell Lineages ◽  
Qrt Pcr ◽  
Nanocomposite Scaffolds

Nanocomposite scaffolds combining carbon nanomaterials (CNMs) with a biocompatible matrix are able to favor the neuronal differentiation and growth of a number of cell types, because they mimic neural-tissue nanotopography and/or conductivity. We performed comparative analysis of biomimetic scaffolds with poly-L-lactic acid (PLLA) matrix and three different p-methoxyphenyl functionalized carbon nanofillers, namely, carbon nanotubes (CNTs), carbon nanohorns (CNHs), and reduced graphene oxide (RGO), dispersed at varying concentrations. qRT-PCR analysis of the modulation of neuronal markers in human circulating multipotent cells cultured on nanocomposite scaffolds showed high variability in their expression patterns depending on the scaffolds’ inhomogeneities. Local stimuli variation could result in a multi- to oligopotency shift and commitment towards multiple cell lineages, which was assessed by the qRT-PCR profiling of markers for neural, adipogenic, and myogenic cell lineages. Less conductive scaffolds, i.e., bare poly-L-lactic acid (PLLA)-, CNH-, and RGO-based nanocomposites, appeared to boost the expression of myogenic-lineage marker genes. Moreover, scaffolds are much more effective on early commitment than in subsequent differentiation. This work suggests that biomimetic PLLA carbon-nanomaterial (PLLA-CNM) scaffolds combined with multipotent autologous cells can represent a powerful tool in the regenerative medicine of multiple tissue types, opening the route to next analyses with specific and standardized scaffold features.

scholarly journals Transcriptional Profiling of Methyltransferase Genes during Growth of Methanosarcina mazei on Trimethylamine

2009 ◽  
Vol 191 (16) ◽  
pp. 5108-5115 ◽  
Author(s):  
Christian Krätzer ◽  
Paul Carini ◽  
Raymond Hovey ◽  
Uwe Deppenmeier
Keyword(s):  
Transcriptional Profiling ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Exponential Growth Phase ◽  
Methane Formation ◽  
Rt Pcr ◽  
Transcript Levels ◽  
Methanosarcina Mazei ◽  
Depth Analysis ◽  
Genes Encoding

ABSTRACT The genomic expression patterns of Methanosarcina mazei growing with trimethylamine were measured in comparison to those of cells grown with methanol. We identified a total of 72 genes with either an increased level (49 genes) or a decreased level (23 genes) of mRNA during growth on trimethylamine with methanol-grown cells as the control. Major differences in transcript levels were observed for the mta, mtb, mtt, and mtm genes, which encode enzymes involved in methane formation from methanol and trimethylamine, respectively. Other differences in mRNA abundance were found for genes encoding enzymes involved in isopentenyl pyrophosphate synthesis and in the formation of aromatic amino acids, as well as a number of proteins with unknown functions. The results were verified by in-depth analysis of methyltransferase genes using specific primers for real-time quantitative reverse transcription-PCR (RT-PCR). The monitored transcript levels of genes encoding corrinoid proteins involved in methyl group transfer from methylated C1 compounds (mtaC, mtbC, mttC, and mtmC) indicated increased amounts of mRNA from the mtaBC1, mtaBC2, and mtaBC3 operons in methanol-grown cells, whereas mRNA of the mtb1-mtt1 operon was found in high concentrations during trimethylamine consumption. The genes of the mtb1-mtt1 operon encode methyltransferases that are responsible for sequential demethylation of trimethylamine. The analysis of product formation of trimethylamine-grown cells at different optical densities revealed that large amounts of dimethylamine and monomethylamine were excreted into the medium. The intermediate compounds were consumed only in the very late exponential growth phase. RT-PCR analysis of key genes involved in methanogenesis led to the conclusion that M. mazei is able to adapt to changing trimethylamine concentrations and the consumption of intermediate compounds. Hence, we assume that the organism possesses a regulatory network for optimal substrate utilization.

scholarly journals Reference gene selection for qRT-PCR assays inStellera chamaejasmesubjected to abiotic stresses and hormone treatments based on transcriptome datasets

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4535 ◽  
Author(s):  
Xin Liu ◽  
Huirui Guan ◽  
Min Song ◽  
Yanping Fu ◽  
Xiaomin Han ◽  
...  
Keyword(s):  
Gene Expression ◽  
Internal Control ◽  
Abiotic Stresses ◽  
Target Genes ◽  
Expression Patterns ◽  
Control Measures ◽  
Rapid Expansion ◽  
Pcr Analysis ◽  
Data Normalization ◽  
Qrt Pcr

BackgroundStellera chamaejasmeLinn, an important poisonous plant of the China grassland, is toxic to humans and livestock. The rapid expansion ofS. chamaejasmehas greatly damaged the grassland ecology and, consequently, seriously endangered the development of animal husbandry. To draft efficient prevention and control measures, it has become more urgent to carry out research on its adaptive and expansion mechanisms in different unfavorable habitats at the genetic level. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used technique for studying gene expression at the transcript level; however, qRT-PCR requires reference genes (RGs) as endogenous controls for data normalization and only through appropriate RG selection and qRT-PCR can we guarantee the reliability and robustness of expression studies and RNA-seq data analysis. Unfortunately, little research on the selection of RGs for gene expression data normalization inS. chamaejasmehas been reported.MethodIn this study, 10 candidate RGs namely,18S,60S,CYP,GAPCP1,GAPDH2,EF1B,MDH,SAND,TUA1, andTUA6, were singled out from the transcriptome database ofS. chamaejasme, and their expression stability under three abiotic stresses (drought, cold, and salt) and three hormone treatments (abscisic acid, ABA; gibberellin, GA; ethephon, ETH) were estimated with the programs geNorm, NormFinder, and BestKeeper.ResultOur results showed thatGAPCP1andEF1Bwere the best combination for the three abiotic stresses, whereasTUA6andSAND,TUA1andCYP,GAPDH2and60Swere the best choices for ABA, GA, and ETH treatment, respectively. Moreover,GAPCP1and60Swere assessed to be the best combination for all samples, and18Swas the least stable RG for use as an internal control in all of the experimental subsets. The expression patterns of two target genes (P5CS2andGI) further verified that the RGs that we selected were suitable for gene expression normalization.DiscussionThis work is the first attempt to comprehensively estimate the stability of RGs inS. chamaejasme. Our results provide suitable RGs for high-precision normalization in qRT-PCR analysis, thereby making it more convenient to analyze gene expression under these experimental conditions.

scholarly journals Selection and Validation of Appropriate Reference Genes for qRT-PCR Analysis of Flowering Stages and Different Genotypes of Iris Germanica L.

2021 ◽  
Author(s):  
Yinjie Wang ◽  
Yongxia Zhang ◽  
Qingquan Liu ◽  
Haiying Tong ◽  
Ting Zhang ◽  
...  
Keyword(s):  
Reference Genes ◽  
Molecular Mechanisms ◽  
Gene Selection ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Herbaceous Plant ◽  
Flower Bud ◽  
Perennial Herbaceous Plant ◽  
Qrt Pcr ◽  
Iris Germanica

Abstract Iris germanica L. is a perennial herbaceous plant that has been widely cultivated worldwide and is popular for its elegant and vibrantly colorful flowers. Selection of appropriate reference genes is the prerequisite for accurate normalization of target gene expression by quantitative real-time PCR. However, to date, the most suitable reference genes for flowering stages have not been elucidated in I. germanica. In this study, eight candidate reference genes were examined for the normalization of qRT-PCR in three I. germanica cultivars, and their stability were evaluated by four different algorithms (GeNorm, NormFinder, BestKeeper, and Ref-finder). The results revealed that IgUBC and IgGAPDH were the most stable reference genes in ‘00246’ and ‘Elizabeth’, and IgTUB and IgUBC showed stable expression in ‘2010200’. IgUBC and IgGAPDH were the most stable in all samples, while IgUBQ showed the least stability. Finally, to validate the reliability of the selected reference genes, the expression patterns of IgFT (Flowering Locus T gene) was analyzed and emphasized the importance of appropriate reference gene selection. This work presented the first systematic study of reference genes selection during flower bud development and provided guidance to research of the molecular mechanisms of flowering stages in I. germanica.

scholarly journals Genome-wide Characterization and Expression Analysis of YABBY Gene Family in Three Cultivars of Cucurbita Linn. And Their Response of Salt Stress in Cucurbita Moschata

2020 ◽  
Author(s):  
Jingping Yuan ◽  
Changwei Shen ◽  
Jingjing Xin ◽  
Zhenxia Li ◽  
Xinzheng Li ◽  
...  
Keyword(s):  
Salt Stress ◽  
Abiotic Stress ◽  
Plant Growth ◽  
Gene Family ◽  
Expression Analysis ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Abiotic Stress Response ◽  
Element Analysis ◽  
Qrt Pcr

Abstract BackgroundPlant specific YABBY transcription factors have important biological roles in plant growth and abiotic stress. However, the identification of Cucurbita Linn. YABBY and their response to salt stress have not yet been reported. The gene number, gene distribution on chromosome, gene structure, protein conserved structure, protein motif and the cis-acting element of YABBY in three cultivars of Cucurbita Linn. were analyzed by bioinformatics tools, and their tissue expression patterns and expression profile under salt stress were analyzed.ResultsIn this study, 34 YABBY genes (11 CmoYABBYs in Cucurbita moschata, 12 CmaYABBYs in Cucurbita maxima, and 11 CpeYABBYs in Cucurbita pepo) were identified and they were divided into five subfamilies (YAB1/YAB3, YAB2, INO, CRC and YAB5). YABBYs in the same subfamily usually have similar gene structures (intron-exon distribution) and conserved domains. Chromosomal localization analysis showed that these CmoYABBYs, CmaYABBYs, and CpeYABBYs were unevenly distributed in 8, 9, and 9 chromosomes of 21 chromosomes, respectively. Total of 6 duplicated gene pairs, and they all experienced segmental duplication events. Cis-acting element analysis showed that some Cucurbita Linn. YABBYs were associated with at least one of plant hormone response, plant growth, and abiotic stress response. Transcriptional profiles of CmoYABBYs and CmaYABBYs in roots, stems, leaves, and fruits, and CpeYABBYs in seed and fruit mesocarp showed that YABBYs of Cucurbita Linn. had tissue specificity. Finally, the transcriptional profile of 11 CmoYABBYs in leaf and qRT-PCR analysis of CmoYABBYs in root under salt stress indicated that some genes may play an important role in salt stress.ConclusionsGenome-wide identification and expression analysis of YABBYs revealed the characteristics of YABBY gene family in three cultivars of Cucurbita Linn.. Transcriptome and qRT-PCR analysis revealed the response of the CmoYABBYs to salt stress.This provides a theoretical basis for the functional research and utilization of YABBY genes in Cucurbita Linn..

scholarly journals Selection and Validation of Reference Genes for qRT-PCR Analysis in the Oil-Rich Tuber Crop Tiger Nut (Cyperus esculentus) Based on Transcriptome Data

2021 ◽  
Vol 22 (5) ◽  
pp. 2569
Author(s):  
Xue Bai ◽  
Tao Chen ◽  
Yuan Wu ◽  
Mingyong Tang ◽  
Zeng-Fu Xu
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Cyperus Esculentus ◽  
Transcriptome Data ◽  
Qrt Pcr ◽  
Below Ground ◽  
The Stability

Tiger nut (Cyperus esculentus), a perennial C4 plant of the Cyperaceae family, is an unconventional crop that is distinguished by its oil-rich tubers, which also possesses the advantages of strong resistance, wide adaptability, short life periods, and large biomass. To facilitate studies on gene expression in this species, we identified and validated a series of reference genes (RGs) based on transcriptome data, which can be employed as internal controls for qRT-PCR analysis in tiger nut. Fourteen putative candidate RGs were identified and evaluated across nine different tissues of two cultivars, and the RGs were analyzed using three different algorithms (geNorm, NormFinder, and BestKeeper). The stability rankings of the candidate RGs were merged into consensus lists with RankAggreg. For the below-ground storage organ of tiger nut, the optimal RGs were TUB4 and UCE2 in different developmental stages of tubers. UCE2 and UBL5 were the most stably expressed RGs among all tissues, while Rubisco and PGK exhibited the lowest expression stability. UCE2, UBL5 and Rubisco were compared to normalize the expression levels of the caleosin (CLO) and diacylglycerol acyltransferase 2-2 (DGAT2-2) genes across the same tissues. Our results showed that the RGs identified in this study, which exhibit more uniform expression patterns, may be utilized for the normalization of qRT-PCR results, promoting further research on gene expression in various tissues of tiger nut.

scholarly journals MicroRNA expression profiles in peri-miniscrew implant crevicular fluid in orthodontics: a pilot study

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Wendan He ◽  
Yanru Yang ◽  
Longgan Cai ◽  
Qiaoling Lei ◽  
Zhongdong Wang ◽  
...  
Keyword(s):  
Mirna Expression ◽  
Kegg Pathway ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Miniscrew Implant ◽  
Pathway Analyses ◽  
Crevicular Fluid

Abstract Background This study systematically evaluated microRNA (miRNA) expression patterns in peri-miniscrew implant crevicular fluid (PMICF) in orthodontic patients. Methods Next-generation sequencing (NGS) was performed to obtain miRNA profiles in PMICF or gingival crevicular fluid (GCF) collected from 3 healthy volunteers (H), 3 peri-implantitis patients (PMSII) and 5 periodontitis patients (P). MiRNA expression patterns were compared between normal and orthodontic PMICF and GCF. Differentially expressed miRNAs were estimated by quantitative real-time PCR (qRT-PCR). Enrichment analyses of the gene targets controlled by these miRNAs were conducted by Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Results Compared with healthy donors, in PMSII patients, a total of 206 upregulated miRNAs and 152 downregulated miRNAs were detected in PMICF, while periodontitis patients had 333 upregulated miRNAs and 318 downregulated miRNAs. MiR-544a, miR-1245b-3p, miR-1825, miR-4291, miR-3689e, and miR-4477a were chosen randomly for further examination. qRT-PCR examination confirmed that the expression levels of miR-1245b-3p and miR-4291 were higher in PMSII than in H samples and that the expression levels of miR-1825 were higher in PMSII than in P samples. However, contrary to the NGS results, qRT-PCR analysis showed decreased expression of miR544a in PMSII. MiR3689e and miR4477a expression did not differ significantly among all samples. According to GO and KEGG pathway analyses of miR-1825, miR-4291, and miR-1245b-3p high enrichment of target genes involved in the PI3K-AKT signalling pathway was observed. Conclusions The NGS analysis of normal and orthodontic PMICF/CGF showed different miRNA profiles, which may lay the foundation for future research on the molecular mechanism of PMSII. miR-4291, miR-1245b-3p and miR-1825 may be used as diagnostic markers and potential therapeutic targets for PMSII.

scholarly journals Activation of the Host Immune Response in Hyphantria cunea (Drury) (Lepidoptera: Noctuidae) Induced by Serratia marcescens Bizio

Insects ◽  
2021 ◽  
Vol 12 (11) ◽  
pp. 983
Author(s):  
Zhiqiang Wang ◽  
Kai Feng ◽  
Fang Tang ◽  
Meng Xu
Keyword(s):  
Immune Response ◽  
Serratia Marcescens ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Hyphantria Cunea ◽  
Immune Genes ◽  
Qrt Pcr ◽  
Cecropin A ◽  
Apoptosis Pathways ◽  
New Ideas

Host–pathogen interactions are essential to our understanding of biological pesticides. Hyphantria cunea (Drury) is an important forest pest worldwide. The immune mechanism of the interaction between H. cunea and Serratia marcescens Bizio (SM1) is unclear. First, transcriptome sequencing and quantitative real-time PCR (qRT-PCR) analysis described the H. cunea immune response to SM1. A total of 234 immune-related differentially expressed genes (DEGs) were found. Many immune regulatory genes in three classical pathways were found. Antimicrobial peptides, including attacin B, cecropin A, gloverin, lebocin and diapausin, are involved in defending against SM1 challenge, and are mainly produced by Toll and immune deficiency (IMD) pathways. Some melanization genes were changed in H. cunea, which suggested that H. cunea melanization was activated by SM1. Furthermore, phagocytosis, autophagolysosome and apoptosis pathways in cellular immunity were activated in H. cunea against SM1. Finally, the expression patterns of 10 immune genes were analyzed systematically by qRT-PCR, and most of the genes were upregulated compared to the control. Our studies provide useful information about the immune response of H. cunea under the stress of SM1, which is important to understand how SM1 affects the immune system of H. cunea and provides new ideas to control H. cunea by using SM1.

scholarly journals Compatative Transcriptome Analysis Reveals Sesquiterpene Biosynthesis among 1-, 2- and 3-year Old Atractylodes Chinensis

2021 ◽  
Author(s):  
Jianhua Zhao ◽  
Chengzhen Sun ◽  
Shanshan Ma ◽  
Jinshuang Zheng ◽  
Xin Du ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Transcriptome Analysis ◽  
High Performance ◽  
Molecular Mechanisms ◽  
Expression Patterns ◽  
Bioactive Compound ◽  
Pcr Analysis ◽  
Squalene Epoxidase ◽  
Sequencing Analysis ◽  
Qrt Pcr

Abstract Background: Atractylodes chinensis (DC.) Koidz is a well-known medicinal plant containing the major bioactive compound, atrctylodin, a sesquiterpenoids. High-performance liquid chromatography (HPLC) analysis demonstrated that atrctylodin was most abundant in 3-year old A. chinensis rhizomes, compared with those from 1-year and 2-year-old plants, however, the molecular mechanisms underlying accumulation of atrctylodin in rhizomes are poorly understood. Results: In this study, we characterized the transcriptomes from 1-, 2, and 3-year old (Y1, Y2, and Y3, respectively) A. chinensis, to identify differentially expressed genes (DEGs). We identified 205 and 226 unigenes encoding the enzyme genes in the mevalonate (MVA) and methylerythritol phosphate (MEP) sesquiterpenoid biosynthesis pathways, respectively. To confirm the reliability of the RNA sequencing analysis, eleven genes key genes encoding factors involved in the sesquiterpene biosynthetic pathway, as well as in pigment, amino acid, hormone, and transcription factor functions, were selected for quantitative real time PCR (qRT-PCR) analysis. The results demonstrated similar expression patterns to those determined by RNA sequencing, with a Pearson’s correlation coefficient of 0.9 between qRT-PCR and RNA-seq data. Differential gene expression analysis of samples from different ages revealed 52 genes related to sesquiterpenoids biosynthesis. Among these, seven DEGs were identified in Y1 vs Y2, Y1 vs Y3, and Y2 vs Y3, of which five encoded four key enzymes, squalene/phytoene synthase, squalene-hopene cyclase, squalene epoxidase and dammarenediol II synthase. These four enzymes directly related to squalene biosynthesis and subsequent catalytic action. To validate the result of these seven DEGs, qRT-PCR was performed and indicated most of them displayed lower relative expression in 3-year old rhizome, similar to transcriptomic analysis. Conclusion: The enzymes SS, SHC, SE and DS down-regulated expression in 3-year old rhizome. This data corresponded to the higher content of sesquiterpenes in 3-year old rhizome, and confirmed by qRT-PCR. The results of comparative transcriptome analysis and identified key enzyme genes laid a solid foundation for investigation of production sesquiterpenoid in A. chinensis.

Metabolomic Analysis of SCD During Goose Follicular Development: Implications for Lipid Metabolism

2020 ◽  
Author(s):  
Xin Yuan ◽  
Shenqiang Hu ◽  
Liang Li ◽  
Hehe Liu ◽  
Hua He ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Expression Patterns ◽  
Follicular Development ◽  
Pcr Analysis ◽  
Qrt Pcr ◽  
Preovulatory Follicles ◽  
Liquid Chromatography Tandem Mass

Abstract Background Stearoyl-CoA desaturase (SCD) is known to be an important rate-limiting enzyme in the production of MUFA. The role of this enzyme in goose follicular development is poorly understood. To investigate the metabolic mechanism of SCD during goose follicular development, we observed SCD expression patterns during follicular development in vivo and in vitro using quantitative reverse-transcription (qRT)-PCR. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine a cellular model of SCD function in granulosa cells (GCs) via SCD overexpression and knockdown.Results qRT-PCR analysis showed that SCD was abundantly expressed in the GC layer, and was upregulated in preovulatory follicles. Peak expression was found in F1 and prehierarchal follicles with diameters of 4–6 mm and 8–10 mm, respectively. We further found the mRNA expression and corresponding enzyme activity to occur in a time-dependent oscillation in vitro, beginning on the first day of GC culture. By using LC-MS/MS, we identified numerous changes in metabolite activation and developed an overview of multiple metabolic pathways, ten of which were associated with lipid metabolism and enriched in both the overexpressed and the knockdown groups.ConclusionsWe confirmed cholesterol and pantothenol or pantothenate as potential metabolite biomarkers for studying SCD-related lipid metabolism in goose GCs.

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