Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse ES cells to generate expression data for validation of single cell cDNA amplification method (V1V3 method)

ABSTRACT: A systems-level understanding of a small but essential population of cells in development or adulthood (e.g., somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the representation of gene expression profiles and reproducibility between individual experiments are unambiguously improved from the original method, along with high coverage and accuracy. Experiment Overall Design: To verify the ability of the developed method, we prepared total RNA from ES cells and diluted it to the single-cell level (10 pg). Eight array data from the cDNA samples amplified independently from the 10 pg RNA (amplified samples) were compared to each other, and to eight array data from the undiluted 5 ug RNA (nonamplified controls). Since all the materials were the same, all of the observed variations were attributable to the methodogoly.

ORGANISM(S): Mus musculus

SUBMITTER: Kazuki Kurimoto

PROVIDER: E-GEOD-4308 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis.

Kurimoto Kazuki K Yabuta Yukihiro Y Ohinata Yasuhide Y Ono Yukiko Y Uno Kenichiro D KD Yamada Rikuhiro G RG Ueda Hiroki R HR Saitou Mitinori M

Nucleic acids research 20060317 5

A systems-level understanding of a small but essential population of cells in development or adulthood (e.g. somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the repr ...[more]

PMID: 16547197

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Project description:An 8-week high fat palm oil diet causes obesity and insulin resistance in C57BL/6J mice, but induces only small changes in the muscle transcriptome. Introduction: The metabolic syndrome (MS) is a cluster of metabolic abnormalities linked to an increased risk of type 2 diabetes and cardiovascular diseases. Two major characteristics of the MS are obesity and insulin resistance. In the present study we investigated the effect of obesity and insulin resistance on the mouse muscle transcriptome. Methods: C57BL/6J mice were fed a palm oil-based low fat diet (LFD) or high fat diet (HFD) for eight weeks. Microarray analysis was performed by using two complementary strategies: (1) 8-week HFD transcriptome versus 8-week LFD transcriptome and (2) transcriptome of mice sacrificed at the start of the intervention versus 8-week LFD transcriptome and 8-week HFD transcriptome, respectively. Results: HFD mice develop obesity and whole-body insulin resistance. Despite these metabolic disturbances we found that HFD induces relatively small changes in gene expression (< 1.3 fold). Only the up-regulation of FA oxidation and the down-regulation of the MAPK cascade were specific for the HFD intervention. Eight-weeks of aging induced more changed gene expression levels than the HFD, including genes involved in cell-cell interaction and development. Conclusion: Eight weeks of aging induce more pronounced changes in the muscle transcriptome than an HFD. Since only one strategy revealed the transcriptional down-regulation of the MAPK cascade, whereas both strategies showed the up-regulation of FA oxidation we suggest the use of complementary analysis strategies by the genome-wide search of gene expression changes induced by mild interventions, such as an HFD. Keywords: diet intervention and time course In this study, C57BL/6J mice were fed a high fat (HF) diet for 8 weeks to induce obesity and insulin resistance. Plasma levels of the adipokines leptin and adiponectin were measured. To investigate how these metabolic disturbances will influence the skeletal muscle transcriptome we performed whole-genome microarray analysis. Functional implications were assessed by analyses of predefined gene sets based on Gene Ontology, biochemical, metabolic and signaling pathways.

Project description:The generation of properly functioning gametes in vitro, a key goal in developmental/reproductive biology, requires multi-step reconstitutions of complex germ cell development. Based on the logic of primordial germ cell (PGC)-specification, we demonstrate here the generation of PGC-like cells (PGCLCs) in mice with robust capacity for spermatogenesis from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) through epiblast-like cells (EpiLCs), a cellular state highly similar to pre-gastrulating epiblasts, but distinct from epiblast stem cells (EpiSCs). The global transcription profiles, epigenetic reprogramming, and cellular dynamics during PGCLC induction from EpiLCs are a meticulous capture of those associated with PGC specification from the epiblasts. Furthermore, we identify Integrin-beta 3 and SSEA1 as markers that purify PGCLCs with spermatogenic capacity free from tumorigenic undifferentiated cells. With the reconstitution of PGC specification pathway from the naive inner cell mass state, our study defines a paradigm for the essential step of in vitro gametogenesis. We performed this analysis to reveal the characters of the cells that we created in this study, epiblast-like cells (EpiLCs) and primordial germ cells-like cells (PGCLCs). Because EpiLCs were induced from embryonic stem cells (ESCs), and equivalent to pre-gastrulating epiblast (embryonic day [E] 5.5-6.0) in vivo (embryo), ESCs and epiblast were included in this analysis. Epiblast stem cells (EpiSCs) are a culture cell type derived from epiblast, and were also included. PGCLCs were supposed to be equivalent to E9.5 PGCs based on reporter fluorescent transgene expressions and epigenetic properties, and therefore E9.5 PGCs were also inckuded in this analysis. Because epiblast and E9.5 PGCs are of a small number of cells in embryos (a few hundred to thousand cells), cDNAs were amplified with a quantitative global PCR method (Kurimoto et al., 2006, Nucleic Acids Research) for microarray analyses. We took two biological replicate for each cell type.

Dataset Information

Transcription profiling of mouse ES cells to generate expression data for validation of single cell cDNA amplification method (V1V3 method)

Publications

An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis.

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