Abstract
Gene expression profiling is an important strategy to study animal development, response to stimuli and diseases. RNAs measured in gene expression profiling experiments are frequently purified from mixture of multiple cell types. The resultant data have low resolution, incapable of distinguishing transcriptome of different cell types and likely biased towards up-regulated genes in dominant tissues. These problems can be solved by obtaining tissue-specific gene expression profile. For dozens of years, there have been several strategies developed to isolate specific tissues or purify RNAs from tissue of interest, and combined with high-throughput RNA assays to generate transcriptome of various specific tissues or cell types. This review will introduce basic principles of these methods and their application in large-scale transcriptome analysis, and discuss on their advantages and limitation.
Highlights
In multicellular organisms, vital processes including development, physiology and response to stimuli require precise regulation of genome on the molecular level, which is manifested as temporal and spatial expression patterns of genes
Splicing based RNA Tagging (SRT) is based on full study in Splicing-Leader-guided trans-splicing, TU-tagging depends on the discovery of uracil phosphoribosyltransferase (UPRT) and PAB-RNA immunoprecipitation (RIP) relies on the discovery of PAB and development in the immunoprecipitation assay
Together with advances in imaging, statistical methods and computational approaches, study of gene expression profiling will guided us to a deeper understanding of gene regulation
Summary
Vital processes including development, physiology and response to stimuli require precise regulation of genome on the molecular level, which is manifested as temporal and spatial expression patterns of genes. For samples that are dissociated, cells can be labelled with fluorescence using fluorescent protein reporter assay or immunofluorescence targeting tissue-specific antigens on cell membranes before fluorescence-activated cell sorting (FACS) is used to collect fluorescence labelled cells for further gene expression profiling. RIP assay targeting poly(A)-binding protein (PAB) driven by tissue-specific promoter can achieve the collection of messenger RNAs from a certain group of cells. This method called PAB-RIP was first tested in C. elegans muscle [26]. PAB-RIP generates large scale tissue-specific transcriptome data, which establishes the foundation of high-throughput and precise gene expression profile. Even in C. elegans, there are ~30% of genes that are not trans-spliced under the mechanism and can not be profiled
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