Project description:Single-cell DNA methylation sequencing is a powerful method for elucidating important physiological and pathological processes, identifying cell subpopulations, and constructing epigenetic regulatory networks. Existing methylome analyses typically require substantial starting materials, complex operations, and high cost and are susceptible to contamination. These problems have impeded the development of single-cell methylome technology for rare cell profiling. In this work, we report Digital Microfluidics-based single-cell Reduced Representation Bisulfite Sequencing (Digital-scRRBS), the first microfluidics-based single-cell methylome library construction platform, which is an automatic, efficient, reproducible, and reagent-economy approach to dissect the single-cell methylome. Taking advantage of our uniquely designed digital microfluidic chip, we realized efficient single-cell isolation in less than 15 seconds. Furthermore, with the advantages of a confined environment, superhydrophobic surface, and nano-scale reaction volume of our digital microfluidic chip, more amplifiable DNA is retained for library construction compared to other approaches. We have successfully constructed single-cell methylation sequencing libraries with a unique genome mapping rate of up to 53.6%, covering up to 2.26 million CpG sites. The application of Digital-scRRBS allows us to discriminate cell identity and dynamically monitor DNA methylation levels during drug administration. Digital-scRRBS provides the technology for widespread application of single-cell methylation methods as a versatile tool for epigenetic analysis in rare cells and highly heterogeneous populations.
Project description:Mild cognitive impairment (MCI) is considered an early stage leading to dementia. MCI can be reversed, and early diagnosis at the MCI stage is vital to control the progression to dementia. Dementia is currently diagnosed based on interviews and screening tests. However, novel biomarkers must be identified to enable early detection of MCI. Therefore, this study aimed to discover novel biomarkers in the form of blood microRNAs (miRNAs) for the diagnosis of MCI or early dementia.
Project description:Background: The diagnosis of Parkinson’s disease (PD) is usually not established until advanced neurodegeneration leads to clinically detectable symptoms. Previous blood PD transcriptome studies show low concordance, possibly due to the use of microarray technology, which has high measurement variation. The Leucine-rich repeat kinase 2 (LRRK2) G2019S mutation predisposes to PD. Using preclinical and clinical studies, we sought to develop a novel statistically motivated transcriptomic-based approach to identify a molecular signature in the blood of Ashkenazi Jewish PD patients including LRRK2 mutation carriers. Methods: Using a digital gene expression platform to quantify 175 mRNA markers with low coefficients of variation (CV), we first compared whole blood transcript levels in mouse models 1) over-expressing wild-type (WT) LRRK2, 2) overexpressing G2019S LRRK2, 3) lacking LRRK2 (knockout), 4) and in WT controls. We then studied an Ashkenazi Jewish cohort of 34 symptomatic PD patients (both WT LRRK2 and G2019S LRRK2) and 32 asymptomatic controls. Results: The expression profiles distinguished the 4 mouse groups with different genetic background. In patients, we detected significant differences in blood transcript levels both between individuals differing in LRRK2 genotype and between PD patients and controls. Discriminatory PD markers included genes associated with innate and adaptive immunity and inflammatory disease. Notably, gene expression patterns in L-DOPA-treated PD patients were significantly closer to those of healthy controls in a dose-dependent manner. Conclusions: We identify whole blood low CV mRNA signatures correlating with LRRK2 genotype and with PD disease state. This approach may provide insight into pathogenesis and a route to early disease detection.
Project description:Background: The diagnosis of Parkinson’s disease (PD) is usually not established until advanced neurodegeneration leads to clinically detectable symptoms. Previous blood PD transcriptome studies show low concordance, possibly due to the use of microarray technology, which has high measurement variation. The Leucine-rich repeat kinase 2 (LRRK2) G2019S mutation predisposes to PD. Using preclinical and clinical studies, we sought to develop a novel statistically motivated transcriptomic-based approach to identify a molecular signature in the blood of Ashkenazi Jewish PD patients including LRRK2 mutation carriers. Methods: Using a digital gene expression platform to quantify 175 mRNA markers with low coefficients of variation (CV), we first compared whole blood transcript levels in mouse models 1) over-expressing wild-type (WT) LRRK2, 2) overexpressing G2019S LRRK2, 3) lacking LRRK2 (knockout), 4) and in WT controls. We then studied an Ashkenazi Jewish cohort of 34 symptomatic PD patients (both WT LRRK2 and G2019S LRRK2) and 32 asymptomatic controls. Results: The expression profiles distinguished the 4 mouse groups with different genetic background. In patients, we detected significant differences in blood transcript levels both between individuals differing in LRRK2 genotype and between PD patients and controls. Discriminatory PD markers included genes associated with innate and adaptive immunity and inflammatory disease. Notably, gene expression patterns in L-DOPA-treated PD patients were significantly closer to those of healthy controls in a dose-dependent manner. Conclusions: We identify whole blood low CV mRNA signatures correlating with LRRK2 genotype and with PD disease state. This approach may provide insight into pathogenesis and a route to early disease detection.
Project description:Rapid and reliable identification of the vertebrate species on which a disease vector previously parasitized is imperative to study ecological factors that affect pathogen distribution and can aid the development of public health programs. We here describe a proteome profiling technique designed to identify the source of blood meals of hematophagous arthropods. This method employs direct spectral matching and thus does not require a priori knowledge of any genetic or protein sequence information. Using this technology, we can detect remnants of blood in blacklegged ticks (Ixodes scapularis) and correctly determine the vertebrate species from which the blood was derived even six months after the tick had fed. This biological fingerprinting methodology is sensitive, fast, cost-effective, and can potentially be adapted for other biological and medical applications when existing genome-based methods are impractical or ineffective.
2014-04-03 | PXD000170 | Pride
Project description:Public description (Provide a description of the study goals and relevance)
Project description:The purpose of this study are: - To identify new biomarkers specific for prostate cancer (PCa) that can be used as diagnostic markers in the urine of individuals with high probability of PCa (abnormal PSA and/or digital rectal examination). - To validate the utility of these new biomarkers, as well as others already known such as PCA3, fusion gene TMPRSS2-ERG, GOLPH2 and SPINK1. - To establish a prediction model for the diagnosis of PCa based on the expression of these biomarkers. - To validate this model within the framework of an opportunist programme of early diagnosis. - Within the framework of this programme, to associate a series of social-demographic, antropometric, life-style and occupational variables for establishing a risk model predictor that could be associated with the model based on the biomarkers.
Project description:We retrospectively analysed the expression of 579 immunological genes in 60 COVID-19 subjects (SARS +ve) and 59 COVID-negative (SARS -ve) subjects using the NanoString nCounter (Immunology panel), a technology based on multiplexed single-molecule counting. Biobanked Human peripheral blood mononuclear cells (PBMCs) samples underwent Nucleic Acid extraction and digital detection of mRNA to evaluate changes in antiviral gene expression between SARS -ve controls and patients with mild (SARS +ve Mild) and moderate/severe (SARS +ve Mod/Sev) disease.
Project description:The purpose of this study are: - To identify new biomarkers specific for prostate cancer (PCa) that can be used as diagnostic markers in the urine of individuals with high probability of PCa (abnormal PSA and/or digital rectal examination). - To validate the utility of these new biomarkers, as well as others already known such as PCA3, fusion gene TMPRSS2-ERG, GOLPH2 and SPINK1. - To establish a prediction model for the diagnosis of PCa based on the expression of these biomarkers. - To validate this model within the framework of an opportunist programme of early diagnosis. - Within the framework of this programme, to associate a series of social-demographic, antropometric, life-style and occupational variables for establishing a risk model predictor that could be associated with the model based on the biomarkers. Methodology: The study is divided in three phases based on three different cohorts of patients: -Phase 1. Identification of biomarkers. This phase includes 60 patients (10 normal prostates and 50 PCa). Form these tumors a screening of miRNAs will be performed. -Phase 2. Validation of the new biomarkers and others already known by means qRT-PCR on urine samples from 300 patients (200 with histological diagnosis of PCa and 100 with an histological negative result). -Phase 3. Prospective validation in a prospective cohort iof 1065 patients included in an opportunist programme of early detection of PCa.