Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform ...transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole-transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2 M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis.
Both the composition of cell types and their spatial distribution in a tissue play a critical role in cellular function, organ development, and disease progression. For example, intratumor ...heterogeneity and the distribution of transcriptional and genetic events in single cells drive the genesis and development of cancer. However, it can be challenging to fully characterize the molecular profile of cells in a tissue with high spatial resolution because microscopy has limited ability to extract comprehensive genomic information, and the spatial resolution of genomic techniques tends to be limited by dissection. There is a growing need for tools that can be used to explore the relationship between histological features, gene expression patterns, and spatially correlated genomic alterations in healthy and diseased tissue samples. Here, we present a technique that combines label-free histology with spatially resolved multiomics in unfixed and unstained tissue sections. This approach leverages stimulated Raman scattering microscopy to provide chemical contrast that reveals histological tissue architecture, allowing for high-resolution in situ laser microdissection of regions of interests. These microtissue samples are then processed for DNA and RNA sequencing to identify unique genetic profiles that correspond to distinct anatomical regions. We demonstrate the capabilities of this technique by mapping gene expression and copy number alterations to histologically defined regions in human oral squamous cell carcinoma (OSCC). Our approach provides complementary insights in tumorigenesis and offers an integrative tool for macroscale cancer tissues with spatial multiomics assessments.
Despite being a relatively recent technological development, single-cell transcriptional analysis through high-throughput sequencing has already been used in hundreds of fruitful studies to make ...exciting new biological discoveries that would otherwise be challenging or even impossible. Consequently, this has fueled a virtuous cycle of even greater interest in the field and compelled development of further improved technical methodologies and approaches. Thanks to the combined efforts of the research community, including the fields of biochemistry and molecular biology, technology and instrumentation, data science, computational biology, and bioinformatics, the single-cell RNA-sequencing field is advancing at a pace that is both astounding and unprecedented. In this review, we provide a broad introduction to this revolutionary technology by presenting the state-of-the-art in sample preparation methodologies, technology platforms, and computational analysis methods, while highlighting the key considerations for designing, executing, and interpreting a study using single-cell RNA sequencing.
An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit ...associated with Huntington's disease, in vitro. Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation.
Microfluidic circuits are characterized by fluidic channels and chambers with a linear dimension on the order of tens to hundreds of micrometers. Components of this size enable lab-on-a-chip ...technology that has much promise, for example, in the development of point-of-care diagnostics. Micro-scale fluidic circuits also yield practical, physical, and technological advantages for studying biological systems, enhancing the ability of researchers to make more precise quantitative measurements. Microfluidic technology has thus become a powerful tool in the life science research laboratory over the past decade. Here we focus on chip-in-a-lab applications of microfluidics and survey some examples of how small fluidic components have provided researchers with new tools for life science research.
The combination of next generation sequencing (NGS) and automated liquid handling platforms has led to a revolution in single-cell genomic studies. However, many molecules that are critical to ...understanding the functional roles of cells in a complex tissue or organs, are not directly encoded in the genome, and therefore cannot be profiled with NGS. Lipids, for example, play a critical role in many metabolic processes but cannot be detected by sequencing. Recent developments in quantitative imaging, particularly coherent Raman scattering (CRS) techniques, have produced a suite of tools for studying lipid content in single cells. This article reviews CRS imaging and computational image processing techniques for non-destructive profiling of dynamic changes in lipid composition and spatial distribution at the single-cell level. As quantitative CRS imaging progresses synergistically with microfluidic and microscopic platforms for single-cell genomic analysis, we anticipate that these techniques will bring researchers closer towards combined lipidomic and genomic analysis.
Quantitative single-cell analysis enables the characterization of cellular systems with a level of detail that cannot be achieved with ensemble measurement. In this Feature we explore quantitative ...cellular imaging applications with nonlinear microscopy techniques. We first offer an introductory tutorial on nonlinear optical processes and then survey a range of techniques that have proven to be useful for quantitative live cell imaging without fluorescent labels.