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    Validating transcripts with probes and imaging technology

    The role of chromosome positioning in gene regulation and chromosome stability is fueling a growing interest in technologies that reveal the in situ organization of the genome.

    Among these technologies are chromosome conformation capture (3C) (1) and its several iterations, such as Hi-C (2), which are applied to populations of nuclei to identify chromosomal regions that are in close proximity to each other (3, 4).

    Initial infection leads to a complex progression through multiple viral gene expression programs after which time the virus typically transitions to a highly latent (type 0 latency), immunologically silent state in memory B cells where no viral protein coding genes are expressed.

    An improved understanding of these approaches is also necessary in order to identify new drug targets, as well as to create particular gene expression patterns.

    The pervasive nature of overlapping transcription throughout herpesvirus genomes, however, poses substantial problems in resolving transcript structures using these methods alone.

    We present an approach that combines the unique attributes of Pacific Biosciences Iso-Seq long-read, Illumina short-read and deep CAGE (Cap Analysis of Gene Expression) sequencing to globally resolve polyadenylated isoform structures in replicating Epstein-Barr virus (EBV).

    A variety of protocols have been developed to measure the expression levels of various RNA species, using either fixed-cell or live-cell imaging methods.

    Despite the fact that much of the knowledge about the spatial distribution of RNA originated from fixed-cell imaging, live-cell imaging strategies provided better possibilities along with added capabilities for real-time monitoring of RNA transport into living cells.

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