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A number of non-membranous cellular bodies have been identified in higher eukaryotes, and these bodies contain a specific set of proteins and RNAs that are used to fulfill their functions. The size of these RNA-containing cellular bodies is usually on a submicron scale, making it difficult to observe fine structures using optical microscopy due to the diffraction limitation of visible light. Recently,...
First evidence on gene function and regulation is provided by the cellular expression pattern in complex tissues. However, to understand the activity of a specific gene, it is essential to analyze the regulatory network, which controls the spatio-temporal translation pattern during the entire life span of the transcribed mRNA. To explore mechanisms which control mRNA abundance and localization in...
mRNA positioning in the cell is important for diverse cellular functions and proper development of multicellular organisms. Single-molecule RNA FISH (smFISH) enables quantitative investigation of mRNA localization and abundance at the level of individual molecules in the context of cellular features. Details about spatial mRNA patterning at various times, in different genetic backgrounds, at different...
A key challenge in mammalian biology is to understand how rates of transcription and mRNA degradation jointly shape cellular gene expression. Powerful techniques have been developed for measuring these rates either genome-wide or at the single-molecule level, however these techniques are not applicable to assessment of cells within their native tissue microenvironment. Here we describe a technique...
Methods for the localization of cellular components such as nucleic acids, proteins, cellular vesicles and more, and the localization of microorganisms including viruses, bacteria and fungi have become an important part of any research program in biological sciences that enable the visualization of these components in fixed and live tissues without the need for complex processing steps. The rapid...
Regulation of mRNA and protein expression occurs at many levels, initiated at transcription and followed by mRNA processing, export, localization, translation and mRNA degradation. The ability to study mRNAs in living cells has become a critical tool to study and analyze how the various steps of the gene expression pathway are carried out. Here we describe a detailed protocol for real time fluorescent...
RNA fluorescence in situ hybridization (FISH) is a widely used technique for detecting transcripts in fixed cells and tissues. Many variants of RNA FISH have been proposed to increase signal strength, resolution and target specificity. The current variants of this technique facilitate the detection of the subcellular localization of transcripts at a single molecule level. Among the applications of...
The vast majority of human protein-coding genes contain up to 90% of non-coding sequence in the form of introns that must be removed from the primary transcripts or pre-mRNAs. Diverse forms of mRNAs encoded from a single gene are created by the differential use of splice sites and alternative splicing is rapidly evolving. Although the kinetic properties of splicing are thought to be critical for proofreading...
Flaviviruses include a wide range of important human pathogens delivered by insects or ticks. These viruses have a positive-stranded RNA genome that is replicated in the cytoplasm of the infected cell. The viral RNA genome is the template for transcription by the virally encoded RNA polymerase and for translation of the viral proteins. Furthermore, the double-stranded RNA intermediates of viral replication...
The regulation of RNA transcription is central to cellular function. Changes in gene expression drive differentiation and cellular responses to events such as injury. RNA trafficking can also have a large impact on protein expression and its localization. Thus, the ability to image RNA transcription and trafficking in real time and in living cells is a worthwhile goal that has been difficult to achieve...
In situ hybridization (ISH) has become an invaluable tool for the detection of RNA in cells, tissues and organisms. Due to improvements in target and signal amplification and in probe design remarkable progress has been made concerning sensitivity, specificity and resolution of chromogenic and fluorescent ISH (FISH). These advancements allow for exquisite cellular and sub-cellular resolution and for...
In situ hybridization is the technique by which specific RNA or DNA molecules are detected in cytological preparations. Basically it involves formation of a hybrid molecule between an endogenous single-stranded RNA or DNA in the cell and a complementary single-stranded RNA or DNA probe. In its original form the probe was labeled with 3H and the hybrid was detected by autoradiography. The first successful...
RNA localization in the Xenopus oocyte is responsible for the establishment of polarity during oogenesis as well as the specification of germ layers during embryogenesis. However, the inability to monitor mRNA localization in live vertebrate oocytes has posed a major barrier to understanding the mechanisms driving directional transport. Here we describe a method for imaging MS2 tagged RNA in live...
The ability to visualize RNA in situ is essential to dissect mechanisms for the temporal and spatial regulation of gene expression that drives development. Although considerable attention has been focused on transcriptional control, studies in model organisms like Drosophila have highlighted the importance of post-transcriptional mechanisms - most notably intracellular mRNA localization - in the formation...
Nascent transcripts being copied from specific human genes can be detected using RNA FISH (fluorescence in situ hybridization) with intronic probes, and the distance between two different nascent transcripts is often measured when studying structure–function relationships. Such distance measurements are limited by the resolution of the light microscope. Here we describe methods for measuring these...
Viruses represent an important class of pathogens that have had an enormous impact on the health of the human race. They are extraordinarily diverse; viral particles can range in size from ∼80nm to ∼10μm in length, and contain genomes with RNA or DNA strands. Regardless of their genome type, RNA species are frequently generated as a part of their replication process, and for viruses with RNA genomes,...
Studies of gene expression are typically carried out by a molecular analysis that averages entire populations of cells in culture, or in a tissue. This approach cannot detect cell to cell variability, or collect subcellular information, such as spatial distribution. At the transcriptional level, it is evident that even a robust transcriptional response in ensemble measurements is not uniform among...
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