Single-cell analysis Dotaz Zobrazit nápovědu
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
nestr.
The proposed project builds on our continuous well-established research on chronic lymphocytic leukemia (CLL). In this study, we would like to profound our knowledge to better understand the mechanisms underlying clonal evolution of CLL cells during the disease course. We will employ a challenging approach a single cell analysis (SCA) for detailed characterisation of malignant cells on single cell level with aim to monitor disease progression and to detect the most aggressive subclones of CLL. In particular, we will analyse and compare transcriptomes of thousands of CLL cells from consecutively collected samples of patients suffering from early relapsed/refractory disease (R/R CLL), which is defined by non-response to treatment or relapse within six months after therapy. Thus, R/R CLL represents a highly challenging subtype of disorder with very poor prognosis and deserves further attention. A deeper understanding of molecular mechanisms driving R/R CLL can help to select the best treatment approach, especially from the growing spectrum of targeted therapy.
Navrhovaný projekt staví na dlouhodobém výzkumu naší skupiny v oblasti chronické lymfocytární leukémie (CLL). Projekt si klade za cíl prohloubit naše znalosti a lépe porozumět mechanismům klonální evoluce CLL buněk v průběhu onemocnění. Pro detailní charakterizaci nádorových buněk a sledování agresivních klonů v průběhu progrese onemocnění chceme využít moderní přístup analýzy na úrovni jednotlivých buněk (SCA). Budeme srovnávat transkriptomy tisíců CLL buněk získaných z opakovaných odběrů pacientů s relaps/refrakterní CLL (R/R CLL) což je onemocnění, které neodpovídá na léčbu nebo u něho dochází k progresi/relapsu dříve než za šest měsíců. R/R CLL reprezentuje významnou podskupinu onemocnění, která si zaslouží pozornost vzhledem k špatné prognóze onemocnění. Porozumění molekulárním mechanismům vedoucím k rozvoji R/R CLL umožní výběr nejvhodnější léčebné strategie, zvlášť v souvislosti s rychlým vývojem cílené léčby v poslední době.
- Klíčová slova
- klonální evoluce, chronic lymphocytic leukemia, clonal evolution, Chronická lymfocytární leukémie, single cell analysis, Single cell analýza,
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
Noninvasive metabolic assessment of single cells / David K. Gardner -- Laser capture microdissection for analysis of single cells / Nobuki Nakamura ... Scriven and Caroline Mackie Ogilvie -- Single cell PCR for PGD : methods, strategies, and limitations [et al.] -- Cell-free fetal DNA plasma extraction and real-time PCR quantification / Jill L. Wangh -- Efficient isothermal amplification of the entire genome from single cells / Karen V.
Methods in molecular medicine, ISSN 1543-1894 132
xi, 182 s. : il., tab.
The cell, as a fundamental structural, functional and biological unit, plays an essential role in living organisms. Analysis of the molecular/elemental composition of a single cell is a significant aspect of lipidomic, proteomic, metabolomic and metallomic studies aiming at understanding molecular processes in cells. Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful technique which can help to elucidate the bioeffects of trace metals and their species on cellular metabolism and cell behavior. Numerous ICP-MS-based methods have already been utilized to explore elemental/species profiles. These include, e.g. time-resolved ICP-MS, electrothermal vaporization ICP-MS, laser ablation ICP-MS, chip-based microextraction techniques, HPLC/CE-ICP-MS, elemental tagging, and mass cytometry. All these methods are covered in this short review.
- MeSH
- analýza jednotlivých buněk metody MeSH
- hmotnostní spektrometrie * metody MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints, applications are increasing every year in all fields of biology. Following the trend, there is a tremendous development of tools for single-cell analysis, especially in the RNA sequencing field. Every improvement increases sensitivity and throughput. Collecting a large amount of data also stimulates the development of new approaches for bioinformatic analysis and interpretation. However, the essential requirement for any analysis is the collection of single cells of high quality. The single-cell isolation must be fast, effective, and gentle to maintain the native expression profiles. Classical methods for single-cell isolation are micromanipulation, microdissection, and fluorescence-activated cell sorting (FACS). In the last decade several new and highly efficient approaches have been developed, which not just supplement but may fully replace the traditional ones. These new techniques are based on microfluidic chips, droplets, micro-well plates, and automatic collection of cells using capillaries, magnets, an electric field, or a punching probe. In this review we summarize the current methods and developments in this field. We discuss the advantages of the different commercially available platforms and their applicability, and also provide remarks on future developments.
Circulating tumor cells (CTCs) are rare cells that can be found in the peripheral blood of cancer patients. They have been demonstrated to be useful prognostic markers in many cancer types. Within the last decade various methods have been developed to detect rare cells within a liquid biopsy from a cancer patient. These methods have revealed the phenotypic diversity of CTCs and how they can represent the complement of cells that are found in a tumor. Single-cell proteogenomics has emerged as an all-encompassing next-generation technological approach for CTC research. This allows for the deconstruction of cellular heterogeneity, dynamics of metastatic initiation and progression, and response or resistance to therapeutics in the clinical settings. We take advantage of this opportunity to investigate CTC heterogeneity and understand their full potential in precision medicine.The high-definition single-cell analysis (HD-SCA) workflow combines detection of the entire population of CTCs and rare cancer related cells with single-cell genomic analysis and may therefore provide insight into their subpopulations based on molecular as well as morphological data. In this chapter we describe in detail the protocols from isolation of a candidate cell from a microscopy slide, through whole-genome amplification and library preparation, to CNV analysis of identified cells from the HD-SCA workflow. This process may also be applicable to any platform starting with a standard microscopy slide or isolated cell of interest.
- MeSH
- analýza jednotlivých buněk MeSH
- Bacteria * MeSH
- interakce mikroorganismu a hostitele * MeSH
- Publikační typ
- úvodníky MeSH
Single-cell transcriptomics has emerged as a powerful tool to investigate cells' biological landscape and focus on the expression profile of individual cells. Major advantage of this approach is an analysis of highly complex and heterogeneous cell populations, such as a specific subpopulation of T helper cells that are known to differentiate into distinct subpopulations. The need for distinguishing the specific expression profile is even more important considering the T cell plasticity. However, importantly, the universal pipelines for single-cell analysis are usually not sufficient for every cell type. Here, the aims are to analyze the diversity of T cell phenotypes employing classical in vitro cytokine-mediated differentiation of human T cells isolated from human peripheral blood by single-cell transcriptomic approach with support of labelled antibodies and a comprehensive bioinformatics analysis using combination of Seurat, Nebulosa, GGplot and others. The results showed high expression similarities between Th1 and Th17 phenotype and very distinct Th2 expression profile. In a case of Th2 highly specific marker genes SPINT2, TRIB3 and CST7 were expressed. Overall, our results demonstrate how donor difference, Th plasticity and cell cycle influence the expression profiles of distinct T cell populations. The results could help to better understand the importance of each step of the analysis when working with T cell single-cell data and observe the results in a more practical way by using our analyzed datasets.
Single cells are basic physiological and biological units that can function individually as well as in groups in tissues and organs. It is central to identify, characterize and profile single cells at molecular level to be able to distinguish different kinds, to understand their functions and determine how they interact with each other. During the last decade several technologies for single-cell profiling have been developed and used in various applications, revealing many novel findings. Quantitative PCR (qPCR) is one of the most developed methods for single-cell profiling that can be used to interrogate several analytes, including DNA, RNA and protein. Single-cell qPCR has the potential to become routine methodology but the technique is still challenging, as it involves several experimental steps and few molecules are handled. Here, we discuss technical aspects and provide recommendation for single-cell qPCR analysis. The workflow includes experimental design, sample preparation, single-cell collection, direct lysis, reverse transcription, preamplification, qPCR and data analysis. Detailed reporting and sharing of experimental details and data will promote further development and make validation studies possible. Efforts aiming to standardize single-cell qPCR open up means to move single-cell analysis from specialized research settings to standard research laboratories.
Analysing the chemical content of individual cells has already been proven to reveal unique information on various biological processes. Single-cell analysis provides more accurate and reliable results for biology and medicine than analyses of extracts from cell populations, where a natural heterogeneity is averaged. To meet the requirements in the research of important biologically active molecules, such as caspases, we have developed a miniaturized device for simultaneous analyses of individual cells. A stainless steel body with a carousel holder enables high-sensitivity parallel detections in eight microvials. The holder is mounted in front of a photomultiplier tube with cooled photocathode working in photon counting mode. The detection of active caspase-3/7, central effector caspases in apoptosis, in single cells is based on the bioluminescence chemistry commercially available as Caspase-Glo®3/7 reagent developed by Promega. Individual cells were captured from a culture medium under microscope and transferred by micromanipulator into detection microvial filled with the reagent. As a result of testing, the limits of detection and quantification were determined to be 0.27/0.86 of active caspase-3/7 content in an average apoptotic cell and 0.46/2.92 for non-apoptotic cells. Application potential of this technology in laboratory diagnostics and related medical research is discussed. Graphical abstract Miniaturized device for simultaneous analyses of individual cells.
- MeSH
- analýza jednotlivých buněk přístrojové vybavení metody MeSH
- apoptóza * MeSH
- design vybavení MeSH
- enzymatické testy přístrojové vybavení metody MeSH
- kaspasa 3 analýza metabolismus MeSH
- kaspasa 7 analýza metabolismus MeSH
- kultivované buňky MeSH
- luminiscenční měření přístrojové vybavení metody MeSH
- myši MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Reverse transcription quantitative PCR (RT-qPCR) has delivered significant insights in understanding the gene expression landscape. Thanks to its precision, sensitivity, flexibility, and cost effectiveness, RT-qPCR has also found utility in advanced single-cell analysis. Single-cell RT-qPCR now represents a well-established method, suitable for an efficient screening prior to single-cell RNA sequencing (scRNA-Seq) experiments, or, oppositely, for validation of hypotheses formulated from high-throughput approaches. Here, we aim to provide a comprehensive summary of the scRT-qPCR method by discussing the limitations of single-cell collection methods, describing the importance of reverse transcription, providing recommendations for the preamplification and primer design, and summarizing essential data processing steps. With the detailed protocol attached in the appendix, this tutorial provides a set of guidelines that allow any researcher to perform scRT-qPCR measurements of the highest standard.
- MeSH
- analýza jednotlivých buněk metody normy MeSH
- kvantitativní polymerázová řetězová reakce metody normy MeSH
- lidé MeSH
- reverzní transkripce genetika MeSH
- senzitivita a specificita MeSH
- stanovení celkové genové exprese metody normy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
