Unknown,Transcriptomics,Genomics,Proteomics

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Real-time quantitative PCR analysis of mouse pre-implantation embryos

ABSTRACT: Adult female mice (C57BL/6) were super-ovulated by intraperitoneal hormone injections with the interval of 48 hours between injections. The hormones administered were PMSG followed by hCG (5IU/0.1ml, Sigma-Aldrich). Following the second hormonal treatment, the females were mated with males (1:1, C57BL/6). Zygotes, 2-cell and 4-cell and blastocysts were collected 26, 48 and 52 hours post hCG. Prior to cell collection, the zona pelucida was removed by Tyrode s acid solution. The 2- and 4-cell embryos were then immersed in Trypsin solution (Invitrogen) with RNase inhibitor (1 IU/ul, Clontech) and BSA (50 ug/ul) for the separation of the blastomeres. The separated blastomeres were snap frozen, and maintained at -80 C until processed. Twelve zygotes, 20 2-cell and 9 4-cell embryos were collected for single cell qPCR. In addition, we prepared with a pool of 10 zygotes and a pool of 50 zygotes as positive controls. The cells and pools were subjected to reverse transcription with SuperScript VILO cDNA Synthesis kit (Life Technologies) as recommended by the manufacturer. DELTAgene Assays were custom designed for 94 genes associated with embryonic development or regulation of gene expression, as well as 2 housekeeping genes. The cDNA from cells and pools were subjected to target specific amplification for 20 cycles. Pre-amplified template was diluted and subjected to primer specific amplification with SooFast EvaGreen Supermix (Biorad) on 96.96 array chip and BiomarkHD System (Fluidigm). Relative quantification of gene expression (log2 space) was obtained by subtracting the Ct values from the baseline value of 22. 12 zygotes, 10 2-cell, and 9 4-cell mouse (C57BL/6) embryos were collected and multi-cell embryos were separated into blastomeres

ORGANISM(S): Mus musculus

SUBMITTER: Fernando Biase

PROVIDER: E-GEOD-59892 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:RNA transcripts are distributed non-uniformly in the oocytes of many animals, such that newly-divided embryo cells (blastomeres) inherit distinct transcriptomes following fertilization. In animals such as the frog, Xenopus laevis, programmed transcript regionalization directs early embryonic axis formation and is essential for normal development. However, it is unknown whether such transcriptome asymmetry directs embryogenesis in mammals, or indeed, whether it occurs at all. We here address this by transcript profiling of matching sub-cellular structures and single cells in mouse oocytes and early embryos and analytical strategies exploiting the paired data structure. Spindle samples contained a set of transcripts that was distinguishable from that of the unfertilized metaphase II (mII) oocytes from which each spindle was microsurgically dissected. Immediately following fertilization, cytokinesis produces a 1-cell embryo (zygote) and associated spindle-enriched second polar body (Pb2) whose transcript profiles also differed one from the other, partially reflecting the enrichment of spindle-associated transcripts. Non-uniform transcript distribution within zygotes did not lead to programmed transcriptome asymmetry between the blastomeres of nascent two-cell embryos, or between the second mitotic products of 3-cell embryos. These findings suggest that mammalian oocytes and zygotes exhibit transcript regionalization without subsequent transcriptome asymmetries between respective early mitotic products. This contrasts the situation in Xenopus and places constraints on the ability of maternal transcriptomic prepatterning to prescribe early mammalian development. 16 Zygote vs. Polar body pairs, 10 spindle vs. oocyte pairs, 22 2-cell embryos and 8 3-cell embryos were analysed. Although the raw data are two channel, only the Cy5 signal of each file was analyzed. The Cy5 channel for each gene is normalized to the average Cy5 intensity of the gene across all samples.

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Real-time quantitative PCR analysis of mouse pre-implantation embryos

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