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A Double-Hybridization Approach for the Transcription- and Amplification-Free Detection of Specific mRNA on a Microarray
摘要： A double-hybridization approach was developed for the enzyme-free detection of specific mRNA of a housekeeping gene. Targeted mRNA was immobilized by hybridization to complementary DNA capture probes spotted onto a microarray. A second hybridization step of Cy5-conjugated label DNA to another section of the mRNA enabled specific labeling of the target. Thus, enzymatic artifacts could be avoided by omitting transcription and amplification steps. This manuscript describes the development of capture probe molecules used in the transcription- and amplification-free analysis of RPLP0 mRNA in isolated total RNA. An increase in specific signal was found with increasing length of the target-specific section of capture probes. Unspecific signal comprising spot autofluorescence and unspecific label binding did not correlate with the capture length. An additional spacer between the specific part of the capture probe and the substrate attachment site increased the signal significantly only on a short capture probe of approximately 30 nt length.
关键词： gene expression,enzyme-free,fluorescence microscopy,mRNA detection,microarray
更新于2025-11-21 11:24:58
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Mapping Neurotransmitter Identity in the Whole-Mount <i>Drosophila</i> Brain Using Multiplex High-Throughput Fluorescence <i>in Situ</i> Hybridization
摘要： Identifying the neurotransmitters used by specific neurons is a critical step in understanding the function of neural circuits. However, methods for the consistent and efficient detection of neurotransmitter markers remain limited. Fluorescence in situ hybridization (FISH) enables direct labeling of type-specific mRNA in neurons. Recent advances in FISH allow this technique to be carried out in intact tissue samples such as whole-mount Drosophila melanogaster brains. Here, we present a FISH platform for high-throughput detection of eight common neurotransmitter phenotypes in Drosophila brains. We greatly increase FISH throughput by processing samples mounted on coverslips and optimizing fluorophore choice for each probe to facilitate multiplexing. As application examples, we demonstrate cases of neurotransmitter co-expression, reveal neurotransmitter phenotypes of specific cell types and explore the onset of neurotransmitter expression in the developing optic lobe. Beyond neurotransmitter markers, our protocols can in principle be used for large scale FISH detection of any mRNA in whole-mount fly brains.
关键词： Neurotransmitter,mRNA,Fluorescence in situ hybridization,Gene expression,Drosophila
更新于2025-11-21 11:08:12
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Enhancing lutein productivity of Chlamydomonas sp. via high-intensity light exposure with corresponding carotenogenic genes expression profiles
摘要： The marine microalga Chlamydomonas sp. JSC4 is a potential lutein source with high light tolerance. In this study, light intensity was manipulated to enhance cell growth and lutein production of this microalga. High lutein productivity (5.08 mg/L/d) was achieved under high light irradiation of 625 μmol/m2/s. Further increase in light intensity to 750 μmol/m2/s enhanced the biomass productivity to 1821.51 mg/L/d, but led to a decrease in lutein content. Under high light conditions, most carotenoids and chlorophyll contents decreased, while zeaxanthin and antheraxanthin contents increased. Inspection of gene expression profile shows that the lut1 and zep genes, responsible for lutein synthesis and flow of zeaxanthin into violaxanthin, respectively, were downregulated, while zeaxanthin biosynthesis gene crtZ was upregulated when the microalga was exposed to a high light intensity. This is consistent with the decrease in lutein content and increase in zeaxanthin content under high light exposure.
关键词： Lutein productivity,high light,Chlamydomonas sp.,gene expression
更新于2025-09-23 15:23:52
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A novel photosensitive dual-sensor for simultaneous detection of nucleic acids and small chemical molecules
摘要： Sensors that can rapidly and specifically detect nucleic acids and chemical molecules can revolutionize the diagnosis and treatment of diseases by allowing molecular-level informations to be used during the routine medicines. In this study, we demonstrated a novel dual-sensor that can be used to simultaneously detect any nucleic acids and chemical molecules whose binding aptamers can be found or synthesized. In the developed dual-sensor, the specifically designed PTG (a photosensitive azobenzene derivative carrying one photo-isomerizable azobenzene moiety, one threoninol terminal and one guanidinium terminal) molecules are introduced into the unwinding region of two T7 promoters, and two DNA bubbles are introduced upstream of the two T7 promoters. Without the target, the indicating gene in the dual-tensor would not be expressed since the binding with RNAPs (RNA polymerases) cannot melt the T7 promoter for the indicating gene due to the integration of the DNA double strands via the PTG molecules, manifesting the absence of the target nucleic acid and chemical molecule. While with the presence of the target nucleic acid and/or chemical molecule, the indicating gene would be expressed as the T7 promoter contained in the enlarged DNA bubble can be melted and transcribed by the bound RNAPs as the enlarged DNA bubble can help the separation of the two DNA strands, demonstrating the existence of target nucleic acid and/or chemical molecule.
关键词： Nucleic acids,DNA melting,Gene expression,DNA nanotechnology,Dual-sensor
更新于2025-09-23 15:23:52
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Infrared Spectroscopic Imaging Visualizes a Prognostic Extracellular Matrix-Related Signature in Breast Cancer
摘要： Molecular analysis techniques such as gene expression analysis and proteomics have contributed greatly to our understanding of cancer heterogeneity. In prior studies, gene expression analysis was shown to stratify patient outcome on the basis of tumor-microenvironment associated genes. A specific gene expression profile, referred to as ECM3 (Extracellular Matrix Cluster 3), indicated poorer survival in patients with grade III tumors. In this work, we aimed to visualize the downstream effects of this gene expression profile onto the tissue, thus providing a spatial context to altered gene expression profiles. Using infrared spectroscopic imaging, we identified spectral patterns specific to the ECM3 gene expression profile, achieving a high spectral classification performance of 0.87 as measured by the area under the curve of the receiver operating characteristic curve. On a patient level, we correctly identified 20 out of 22 ECM3 group patients and 19 out of 20 non-ECM3 group patients by using this spectroscopic imaging-based classifier. By comparing pixels that were identified as ECM3 or non-ECM3 with H&E and IHC images, we were also able to observe an association between tissue morphology and the gene expression clusters, showing the ability of our method to capture broad outcome associated features from infrared images.
关键词： extracellular matrix,prognostic signature,gene expression,breast cancer,infrared spectroscopic imaging
更新于2025-09-23 15:21:01
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Combining Laser Capture Microdissection and Microfluidic qPCR to Analyze Transcriptional Profiles of Single Cells: A Systems Biology Approach to Opioid Dependence
摘要： Profound transcriptional heterogeneity in anatomically adjacent single cells suggests that robust tissue functionality may be achieved by cellular phenotype diversity. Single-cell experiments investigating the network dynamics of biological systems demonstrate cellular and tissue responses to various conditions at biologically meaningful resolution. Herein, we explain our methods for gathering single cells from anatomically specific locations and accurately measuring a subset of their gene expression profiles. We combine laser capture microdissection (LCM) with microfluidic reverse transcription quantitative polymerase chain reactions (RT-qPCR). We also use this microfluidic RT-qPCR platform to measure the microbial abundance of gut contents.
关键词： single cell gene expression,amygdala,gut microbiome,anatomic specificity,glia,laser capture microdissection,opioid dependence,Genetics,microfluidic qPCR
更新于2025-09-23 15:21:01
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Stress Physiology of Tea in the Face of Climate Change || UV-B Radiation-Induced Changes in Tea Metabolites and Related Gene Expression
摘要： UV-B radiation is an inevitable abiotic stress, which could induce a series of changes in metabolites and related metabolisms in plants. UV-B-induced metabolic changes in leaves of Camellia sinensis affect the tea quality. This review summarizes the recent investigations into UV-B radiation-induced changes in tea metabolites and their related gene expression, involving in flavonoids, amino acids, and volatile compounds. UV-B radiation induces flavonoid accumulation by increasing expression of key genes in general phenylpropanoid pathway and flavonoid pathway. The UV-B radiation-induced gene expressions in flavonoid biosynthesis pathway also are affected by transcription factors and endogenous phytohormones signaling pathway. Changes of individual amino acids under UV-B radiation exhibit significant variation among different plants, and their responses to UV-B radiation dose are different. These regulations involve in modulation of gene expressions related to GABA shunt and tricarboxylic acid cycle (TCA). Volatile compounds in Camellia sinensis under UV-B radiation are regulated by both metabolites biosynthesis and volatile glycosidic-precursors hydrolysis. In a word, UV-B radiation influences metabolisms in tea in a rather complex way. More researches on UV-B-induced transcriptional regulation, endogenous-phytohormone signal regulation, metabolisms diversions regulation, etc. are needed in the future.
关键词： Gene expression,Signal regulation,Amino acids,Volatile compounds,Tea polyphenols,UV-B radiation
更新于2025-09-23 15:21:01
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Systems toxicology assessment revealed the impact of graphenea??based materials on cell cycle regulators
摘要： Understanding the cellular and molecular toxicity of graphene and its derivatives is essential for their biomedical applications. Herein, gene expression profile of graphene-exposed cells was retrieved from the GEO database. Differentially expressed genes and their functional roles were then investigated through the pathway, protein-protein interaction network, and module analysis. High degree (hub) and high betweenness centrality (bottleneck) nodes were subsequently identified. The functional analysis of central genes indicated that these graphene-gene interactions could be of great value for further investigation. Accordingly, we also followed the expression of five hub-bottleneck genes in graphene-treated murine peritoneal macrophages and human breast cancer cell line by real-time PCR. The five hub-bottleneck genes related to graphene cytotoxicity; CDK1, CCNB1, PLK1, TOP2A, and CCNA2 were identified through network analysis, which were highly correlated with regulation of cell cycle processes. The module analysis indicated the cell cycle pathway to be the predominant one. Gene expression evaluation showed down-regulation of these genes in the macrophages and cancer cells treated with graphene. These results provided some new intuitions concerning the graphene-cell interactions and unveiled targeting critical cell cycle regulators. The present study indicated some toxic effects of graphene-based materials through systems toxicology assessment. Integrating gene expression and protein-protein interaction network may help explaining biological responses of graphene and lead to beneficial impacts in nanomedicine.
关键词： Cell cycle,Graphene,Protein-protein interaction network,Gene expression,Systems toxicology
更新于2025-09-23 15:19:57
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A molecular ferroelectrics induced electroactive β-phase in solution processed PVDF films for flexible piezoelectric sensors
摘要： Background: Precise diagnosis of the tissue origin for metastatic cancer of unknown primary (CUP) is essential for deciding the treatment scheme to improve patients’ prognoses, since the treatment for the metastases is the same as their primary counterparts. The purpose of this study is to identify a robust gene signature that can predict the origin for CUPs. Methods: The within-sample relative gene expression orderings (REOs) of gene pairs within individual samples, which are insensitive to experimental batch effects and data normalizations, were exploited for identifying the prediction signature. Results: Using gene expression profiles of the lung-limited metastatic colorectal cancer (LmCRC), we firstly showed that the within-sample REOs in lung metastases of colorectal cancer (CRC) samples were concordant with the REOs in primary CRC samples rather than with the REOs in primary lung cancer. Based on this phenomenon, we selected five gene pairs with consistent REOs in 498 primary CRC and reversely consistent REOs in 509 lung cancer samples, which were used as a signature for predicting primary sites of metastatic CRC based on the majority voting rule. Applying the signature to 654 primary CRC and 204 primary lung cancer samples collected from multiple datasets, the prediction accuracy reached 99.36%. This signature was also applied to 24 LmCRC samples collected from three datasets produced by different laboratories and the accuracy reached 100%, suggesting that the within-sample REOs in the primary site could reveal the original tissue of metastatic cancers. Conclusions: The result demonstrated that the signature based on within-sample REOs of five gene pairs could exactly and robustly identify the primary sites of CUPs.
关键词： Lung cancer,Cancer of unknown primary,Colorectal cancer,Relative gene expression orderings,Metastasis
更新于2025-09-19 17:15:36
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Investigation of the therapeutic mechanism of subthreshold micropulse laser irradiation in retina
摘要： Purpose Subthreshold micropulse laser irradiation has been used for the treatment of retinal edema; however, there are few reports about the mechanism of its therapeutic effect. In this study, we compared threshold short pulse and subthreshold micropulse laser irradiation in mice and investigated their mechanism. Methods Nine to 12-week-old male C57BL/6J mice were used in this study. After general anesthesia, threshold short pulse or subthreshold micropulse laser irradiation was performed on the right eye using IQ577. Enucleation was performed 24 h after the laser irradiation, and histological and gene expression analyses were carried out. Results Coagulation spots and atrophy of the retinal pigment epithelium were observed after threshold short pulse laser irradiation but not after subthreshold micropulse laser irradiation. Twenty-four hours after laser, aquaporin (AQP) 1, 2, 7, and 11 levels were significantly elevated by 1.7- to 3-fold in the threshold short pulse laser group compared with non-treated control group. AQP 3 was increased significantly and prominently by 100-fold. VEGF-A and VEGFR2 were upregulated 1.5- and 2.3-fold, respectively. In the subthreshold micropulse laser group, AQP 3 was increased by 6-fold compared with the non-treated control group. Angiopoietin-1 and the adrenomedullin (AM) receptor CLR were decreased by 0.6-fold and 0.5-fold, respectively. Conclusion Threshold short pulse laser irradiation caused retinal damage and prominent changes in the expression of various genes. Contrarily, subthreshold micropulse laser irradiation did not induce retinal damage; it upregulated AQP 3, which might have improved retinal edema by drainage of subretinal fluid.
关键词： Retinal edema,Aquaporin-3,Gene expression,Subthreshold micropulse laser
更新于2025-09-19 17:13:59