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Comparing the samples with and without RNA amplification

ABSTRACT: Diverse cell types have unique transcriptional signatures that are best interrogated at single-cell resolution. Here we describe a novel RNA amplification approach that allows for high fidelity gene profiling of individual cells. This technique significantly diminishes the problem of 3’bias and therefore enables detection of all regions of transcripts, including the recognition of mRNA with short or completely absent polyA tails, identification of noncoding RNAs, and discovery of the full array of splice isoforms from any given gene product. We assess this technique using statistical and bioinformatics analysis of microarray data in order to establish the limitations of the method. To demonstrate the applicability of the method, we conducted single cell profiling of individual cells, acutely isolated from the mouse subventricular zone (SVZ) – a well-characterized, discrete yet highly heterogeneous neural structure involved in persistent neurogenesis. Importantly, this method revealed multiple splice variants of key germinal zone gene products, as well as an unexpected coexpression, within individual cells, of several mRNAs that are considered markers of distinct and separate SVZ cell types. These findings were independently confirmed using RNA-FISH, contributing to the utility of this new technology that offers genomic and transcriptomic analysis of small numbers of, in this case, extremely dynamic and clinically relevant cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE55137 | GEO | 2014/02/20

SECONDARY ACCESSION(S): PRJNA238595

REPOSITORIES: GEO

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Project description:Throughout postnatal life in mammals, neural stem cells (NSCs) are located in the subventricular zone (SVZ) of the lateral ventricles. The greatest diversity of neuronal and glial lineages they generate occurs during early postnatal life in a region-specific manner. In order to evaluate potential heterogeneity in the NSC pool, we microdissected the dorsal and lateral SVZ at different postnatal ages and isolated NSCs and their immediate progeny based on their expression of Hes5-EGFP/Prominin1 and Ascl1-EGFP, respectively. Whole genome comparative transcriptome analysis revealed transcriptional regulators as major hallmarks that sustain postnatal SVZ regionalization. Manipulation of single genes encoding for locally enriched transcription factors influenced NSC specification indicating that the fate of regionalized postnatal SVZ NSCs can be readily modified . These findings reveal functional heterogeneity of NSCs in the postnatal SVZ and provide targets to recruit region-specific lineages in regenerative contexts. Microarrays of neural stem cells, early progenitors and the tissue from subregions of the subventricular zone were compiled to screen for the full extent of heterogeneity in this region during postnatal life. Spatially distinct regions of the developing forebrain subventricular zone (SVZ) aged at P4, P8 and P11 were microdissected in RNAse free/sterile conditions. Mice expressing Ascl1-EGFP in the SVZ were used to aid accurate microdissection of the dorsal and lateral wall of each of the studied time points as per our previous publications characterizing this method. As well as at the whole microdomain level, additionally, NSCs (Hes5-EGFP+/Prom1+) and early progenitors (Ascl1-EGFP+) from each microdomain were further isolated by FAC sorting methods. This was to provide a comprehensive gene expression analysis at the tissue level and at the cellular level. Generally, 1 litter was used to yield 1 'n' number of replicates. A total of 23 affymetrix analysis were performed.

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Comparing the samples with and without RNA amplification

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