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.

• Klíčová slova
• MIQE guidelines, Single-cell analysis, Single-cell qPCR, Single-cell workflow, qPCR,
• MeSH
• analýza jednotlivých buněk metody   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• lidé MeSH
• polymerázová řetězová reakce MeSH
• stanovení celkové genové exprese metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy 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.

• Klíčová slova
• RT-qPCR, gene expression, preamplification, quantitative PCR, reverse transcription, sample collection, single cell,
• 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

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.

• Klíčová slova
• analysis, collection, isolation, single cell,
• MeSH
• analýza jednotlivých buněk přístrojové vybavení   metody   MeSH
• lidé MeSH
• mikrofluidika přístrojové vybavení   metody   MeSH
• průtoková cytometrie přístrojové vybavení   metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here, we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR. Four endogenously expressed genes are assayed together with RNA and DNA spikes in the samples. We found bovine serum albumin (BSA) to be the best lysis agent, resulting in efficient cell lysis, high RNA stability, and enhanced reverse transcription efficiency. Furthermore, we found direct cell lysis with BSA superior to standard column based extraction methods, when analyzing from 1 up to 512 mammalian cells. In conclusion, direct cell lysis protocols based on BSA can be applied with most cell collection methods and are compatible with most analytical workflows to analyze single-cells as well as samples composed of small numbers of cells.

• Klíčová slova
• DNA spike, RNA purification, RNA spike, cell lysis, direct lysis, real-time PCR, single-cell biology, single-cell gene expression,
• Publikační typ
• časopisecké články MeSH

Triple-negative breast cancer (TNBC) is an aggressive and complex subtype of breast cancer that lacks targeted therapy. TNBC manifests characteristic, extensive intratumoral heterogeneity that promotes disease progression and influences drug response. Single-cell techniques in combination with next-generation computation provide an unprecedented opportunity to identify molecular events with therapeutic potential. Here, we describe the generation of a comprehensive mass cytometry panel for multiparametric detection of 23 phenotypic markers and 13 signaling molecules. This single-cell proteomic approach allowed us to explore the landscape of TNBC heterogeneity, with particular emphasis on the tumor microenvironment. We prospectively profiled freshly resected tumors from 26 TNBC patients. These tumors contained phenotypically distinct subpopulations of cancer and stromal cells that were associated with the patient's clinical status at the time of surgery. We further classified the epithelial-mesenchymal plasticity of tumor cells, and molecularly defined phenotypically diverse populations of tumor-associated stroma. Furthermore, in a retrospective tissue-microarray TNBC cohort, we showed that the level of CD97 at the time of surgery has prognostic potential.

• Klíčová slova
• mass cytometry, phenotypic plasticity, single-cell profiles, triple-negative breast cancer, tumor heterogeneity, unsupervised machine learning algorithm,
• MeSH
• buňky stromatu metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorové mikroprostředí MeSH
• proteomika MeSH
• retrospektivní studie MeSH
• signální transdukce MeSH
• triple-negativní karcinom prsu * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• CNV, CTC, Circulating tumor cells, Copy number variation, Liquid biopsy, Precision medicine, Single-cell analysis,
• MeSH
• analýza jednotlivých buněk metody   MeSH
• individualizovaná medicína MeSH
• lidé MeSH
• mutace MeSH
• nádorové cirkulující buňky MeSH
• nádory diagnóza   genetika   MeSH
• proteogenomika metody   MeSH
• variabilita počtu kopií segmentů DNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

We lack a holistic understanding of the genetic programs orchestrating embryonic colon morphogenesis and governing damage response in the adult. A window into these programs is the transcriptomes of the epithelial and mesenchymal cell populations in the colon. Performing unbiased single-cell transcriptomic analyses of the developing mouse colon at different embryonic stages (embryonic day 14.5 [E14.5], E15.5, and E18.5), we capture cellular and molecular profiles of the stages before, during, and after the appearance of crypt structures, as well as in a model of adult colitis. The data suggest most adult lineages are established by E18.5. We find embryonic-specific gene expression profiles and cell populations that reappear in response to tissue damage. Comparison of the datasets from mice and human colitis suggests the processes are conserved. In this study, we provide a comprehensive single-cell atlas of the developing mouse colon and evidence for the reactivation of embryonic genes in disease.

• Klíčová slova
• DSS, Single cell RNA sequencing, colitis, colon development, colon inflammatory disease, hindgut development, intestinal damage, intestinal development, single-cell atlas,
• MeSH
• analýza jednotlivých buněk MeSH
• buněčná diferenciace MeSH
• embryo savčí metabolismus   MeSH
• idiopatické střevní záněty genetika   patologie   MeSH
• kolitida genetika   MeSH
• kolon embryologie   patologie   MeSH
• lidé MeSH
• mezoderm embryologie   MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• stanovení celkové genové exprese * MeSH
• střevní sliznice embryologie   metabolismus   patologie   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Osteogenesis is an important process of bone metabolism, and abnormal osteogenesis leads to various skeletal system diseases. Osteoblasts, the main cells involved in bone formation, are central elements in the study of bone metabolic diseases. Single-cell RNA sequencing is an important tool for studying the transcriptome of cells and can help to elucidate various cellular and molecular functions at the single-cell level, providing new avenues for life science research. Here we explore the heterogeneity of osteoblasts and try to reveal the developmental trajectory of osteoblasts, thereby contributing to efforts to describe the mechanism of osteogenesis. In this study, single-cell sequencing data of murine bone marrow cells were used to identify osteoblasts. Finally, osteoblasts were divided into four groups, each differing in characteristic genes and signal pathways. We also identify clues of the changes of some genes in the process of osteoclast formation, providing directions for further study. Collectively, our findings suggest that bone marrow osteoblasts can be divided into several subgroups, which represent different stages of cells, and that the specific genes of each subgroup respond to the molecular mechanisms of cell development. This data will likely be of great help in resolving diseases of the skeletal system.

• MeSH
• buněčná diferenciace MeSH
• kostní dřeň * MeSH
• myši MeSH
• osteoblasty * metabolismus   MeSH
• osteogeneze genetika   MeSH
• osteoklasty metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• Activation, Cell cycle regression, Correction for batch effect, Data analysis, Differential expression, Differentiation, Gene expression profiling, Plasticity, Signature genes, Single-cell RNA sequencing, T helper cells,
• MeSH
• aktivace lymfocytů * MeSH
• buněčná diferenciace genetika   MeSH
• buňky Th17 MeSH
• lidé MeSH
• membránové glykoproteiny metabolismus   MeSH
• sekvenční analýza RNA MeSH
• Th2 buňky * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové glykoproteiny MeSH
• SPINT2 protein, human MeSH Prohlížeč

BACKGROUND: Apicomplexa is a diverse phylum comprising unicellular endobiotic animal parasites and contains some of the most well-studied microbial eukaryotes including the devastating human pathogens Plasmodium falciparum and Cryptosporidium hominis. In contrast, data on the invertebrate-infecting gregarines remains sparse and their evolutionary relationship to other apicomplexans remains obscure. Most apicomplexans retain a highly modified plastid, while their mitochondria remain metabolically conserved. Cryptosporidium spp. inhabit an anaerobic host-gut environment and represent the known exception, having completely lost their plastid while retaining an extremely reduced mitochondrion that has lost its genome. Recent advances in single-cell sequencing have enabled the first broad genome-scale explorations of gregarines, providing evidence of differential plastid retention throughout the group. However, little is known about the retention and metabolic capacity of gregarine mitochondria. RESULTS: Here, we sequenced transcriptomes from five species of gregarines isolated from cockroaches. We combined these data with those from other apicomplexans, performed detailed phylogenomic analyses, and characterized their mitochondrial metabolism. Our results support the placement of Cryptosporidium as the earliest diverging lineage of apicomplexans, which impacts our interpretation of evolutionary events within the phylum. By mapping in silico predictions of core mitochondrial pathways onto our phylogeny, we identified convergently reduced mitochondria. These data show that the electron transport chain has been independently lost three times across the phylum, twice within gregarines. CONCLUSIONS: Apicomplexan lineages show variable functional restructuring of mitochondrial metabolism that appears to have been driven by adaptations to parasitism and anaerobiosis. Our findings indicate that apicomplexans are rife with convergent adaptations, with shared features including morphology, energy metabolism, and intracellularity.

• Klíčová slova
• Anaerobic metabolism, Apicomplexa, Eugregarines, Evolution, Mitochondria, Mitosome, Parasitism, Phylogenomics,
• MeSH
• analýza jednotlivých buněk MeSH
• Apicomplexa * genetika   MeSH
• fylogeneze MeSH
• lidé MeSH
• mitochondrie * genetika   MeSH
• transkriptom MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH