Unknown,Transcriptomics,Genomics,Proteomics

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Database for mRNA Half-Life of 19,977 Genes Obtained by DNA Microarray Analysis of Pluripotent and Differentiating Mouse

ABSTRACT: Degradation of mRNA is one of the key processes that control the steady-state level of gene expression. However, the rate of mRNA decay for the majority of genes is not known. We successfully obtained the rate of mRNA decay for 19 977 non-redundant genes by microarray analysis of RNA samples obtained from mouse embryonic stem (ES) cells. Median estimated half-life was 7.1 h and only <100 genes, including Prdm1, Myc, Gadd45 g, Foxa2, Hes5 and Trib1, showed half-life less than 1 h. In general, mRNA species with short half-life were enriched among genes with regulatory functions (transcription factors), whereas mRNA species with long half-life were enriched among genes related to metabolism and structure (extracellular matrix, cytoskeleton). The stability of mRNAs correlated more significantly with the structural features of genes than the function of genes: mRNA stability showed the most significant positive correlation with the number of exon junctions per open reading frame length, and negative correlation with the presence of PUF-binding motifs and AU-rich elements in 30 -untranslated region (UTR) and CpG di-nucleotides in the 50 -UTR. The mRNA decay rates presented in this report are the largest data set for mammals and the first for ES cells. Keywords: growth condition design,reference design,replicate design,RNA stability design,time series design We used two mouse ESC lines: MC1 derived from mouse strain 129S6/SvEvTac and MC2-B6 derived from strain C57BL/6J. To remove contaminating feeder cells, ES cells were first cultured for two passages on gelatin-coated culture dish in complete ES medium. Differentiated ES cells were obtained from MC1 cells by LIF withdrawal (complete medium without LIF, below referenced as LIF-) or by retinoic acid (RA) treatment. To measure mRNA stability, transcription was blocked by adding actinomycin D to the medium. RNA was hybridized in duplicate for each cell type and time point. All hybridizations were carried out by combining a Cy3-CTP-labeled experimental target and a Cy5-CTP-labeled Universal Mouse Reference target.

ORGANISM(S): Mus musculus

SUBMITTER: Minoru KO

PROVIDER: E-GEOD-13609 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Database for mRNA half-life of 19 977 genes obtained by DNA microarray analysis of pluripotent and differentiating mouse embryonic stem cells.

Sharova Lioudmila V LV Sharov Alexei A AA Nedorezov Timur T Piao Yulan Y Shaik Nabeebi N Ko Minoru S H MS

DNA research : an international journal for rapid publication of reports on genes and genomes 20081111 1

Degradation of mRNA is one of the key processes that control the steady-state level of gene expression. However, the rate of mRNA decay for the majority of genes is not known. We successfully obtained the rate of mRNA decay for 19 977 non-redundant genes by microarray analysis of RNA samples obtained from mouse embryonic stem (ES) cells. Median estimated half-life was 7.1 h and only <100 genes, including Prdm1, Myc, Gadd45 g, Foxa2, Hes5 and Trib1, showed half-life less than 1 h. In general, mRN ...[more]

PMID: 19001483

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Project description:We present a microarray experiment with the same tet-inducible system but with multiple time points within 24 hr to capture early responses to Oct4 suppression. These data were analyzed together with published genome-wide ChIP data. We found that most tentative target genes (TTG) of Oct4 in ES cells were activated by Oct4 and only a few genes were suppressed. The same method was then applied to find target genes of Sox2 and Nanog based mostly on previously published data. Because the interaction between Oct4, Sox2, and Nanog is supported by immunoprecipitation, functional analysis, and co-localization of binding sites, we explored the relationships between their target genes. Keywords: time series design We used ZHBTc4 ES cells with a Tet-inducible Oct4 transgene. Cells were cultured for 2 passages on gelatin-coated plates in order to remove feeder cells and then transferred to gelatin-coated 6-well plates at the density of 1-2x105 cells/well and cultured in complete ES medium. Tetrocycline (TC) was added at 24 hr after cell plating, and then cells were harvested at 0hr (before adding TC), 3 hr, 6 hr, 12 hr, and 24 hr (2 replications each). RNA samples for later time points (24, 48, 72, 96, and 120 hr) were obtained from our earlier experiment with 3 replications. Two Nanog over-expressing clones were tested: (1) integrated transgene in MG1.19 parental cell line and (2) episomal transgene in EBRTcH3 parental line. EBRTcH3 and MG1.19 ESC lines were kind gifts of Dr. Hitoshi Niwa (RIKEN Center for Developmental Biology, Kobe, Japan). Both transgenic and parental cell lines were cultured for 2 passages on gelatin-coated plates and then transferred to gelatin-coated 6-well plates at the density of 1-2x105 cells/well and cultured for 3 days in 3 different conditions: (1) complete ES medium (see above); (2) complete medium without LIF, and (3) complete medium with 1 uM RA. Cells were cultured at 37 0C and 5% CO2 condition and the culture medium was changed daily. Over-expression of Nanog was evaluated by PCR. Total RNAs were extracted using TrizolTM (Invitrogen, USA) and Phase lock gelTM columns (Eppendorf/Brinkman) according to the manufacturer???s protocol. Total RNAs were precipitated with isopropanol, washed with 70% ethanol, and dissolved in DEPC-treated H2O. 2.5 ug of total RNA samples were labeled with Cy3-CTP using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent, USA). A reference target (Cy5-CTP-labeled) was prepared from the Universal Mouse Reference (UMR) RNA (Stratagene, USA).

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Dataset Information

Database for mRNA Half-Life of 19,977 Genes Obtained by DNA Microarray Analysis of Pluripotent and Differentiating Mouse

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Database for mRNA half-life of 19 977 genes obtained by DNA microarray analysis of pluripotent and differentiating mouse embryonic stem cells.

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