Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of Bos taurus immature oocytes and 8-cell embryos to assess the impact of the loss of sample representation caused by the use of global linear amplification (T7-IVT) during microarray hybridization sample preparation

ABSTRACT: Assessing the impact of the loss of sample representation caused by the use of global linear amplification (T7-IVT) during microarray hybridization sample preparation.

ORGANISM(S): Bos taurus

SUBMITTER: Isabelle Gilbert

PROVIDER: E-MEXP-2099 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Real-time monitoring of aRNA production during T7 amplification to prevent the loss of sample representation during microarray hybridization sample preparation.

Gilbert Isabelle I Scantland Sara S Dufort Isabelle I Gordynska Olga O Labbe Aurélie A Sirard Marc-André MA Robert Claude C

Nucleic acids research 20090330 8

Gene expression analysis performed through comparative abundance of transcripts is facing a new challenge with the increasing need to compare samples of known cell number, such as early embryos or laser microbiopsies, where the RNA contents of identical cellular inputs can by nature be variable. When working with scarce tissues, the success of microarray profiling largely depends on the efficiency of the amplification step as determined by its ability to preserve the relative abundance of transc ...[more]

PMID: 19336411

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Project description:Blastocyst formation is a primordial event of pre-implantation development since it is required for pregnancy establishment and progression. The blastocyst plays a pivotal role because it is the stage at which the embryo is transferred and starts coordinated cross-talks with the mother. It is also the terminal step of pre-implantation developmentbefore transfer; it reflects all stresses the embryo may have faced during the process of in-vitro treatment. Achieving the formation of a morphologically healthy blastocyst following normal kinetics is a good sign but remains a poor indicator of embryo quality. Considering the limitation of the invasive methods for competence assessment, the analysis of blastocysts’ gene expression is a promising way to improve our understanding of blastocyst formation and to study the effects of different treatments on gene expression. Methods: Early, expanded and hatched blastocysts were collected for RNA extraction, amplification, and cDNA microarray hybridization. Gene candidates (IFNt, PLAC8, SSLP1, AKR1B1, HNRNPA2B1, ARGFX, NANOS, CCNB1) were selected and confirmed using Q-RT-PCR to validate the microarray data. Results: Our analysis show that hatched blastocysts are enriched in genes transcripts implicated in implantation, cell adhesion and extracellular matrix digestion. Early blastocysts expressed genes mainly involved in cell cycle control, transcription and translation. Q-RT-PCR validated most microarray results (87.5%). Some of the differentially expressed genes are interesting as potential markers of competence. Conclusions: Overall, our study provides new insights into the molecular regulation of blastocyst formation. In addition, it could help assess blastocyst staging and select better embryos based on the expression of quality markers. In the present study the Laval/Sirard_bovine_embryo_3K_V3.0 array was used to conduct a series of 18 hybridizations (for three Blastocysts stages (early, expended, hatched) with three biological replicates and dye swaps) in a loop design experiment.

Project description:In vitro production of cattle embryos over the past two decades has revealed several negative impacts, which have been attributed to the artificial microenvironment. Studies of embryos produced in vitro clearly point to aberrant gene expression levels. So far, the causal association between phenotype and measured gene expression has not led to substantial improvement of in vitro production systems. The aim of the present study was to generate a unique dataset composed of microarray-derived relative transcript abundance values for blastocysts produced in ten in vitro systems differing primarily in culture medium formulation. Between-group comparisons allowed us to determine the level of overall similarity between systems relative to in vivo reference embryos. The use of the dataset to contrast all in vitro treatments with the in vivo blastocysts pointed to a single common gene network. The “boutique” array contained a panel of novel uncharacterized transcripts that were expressed variably depending on the medium in which the blastocysts were produced. These novel transcripts were expressed differentially in blastocysts even as carry-over from conditions encountered seven days earlier during oocyte maturation. All of the selected novel candidates that were thus expressed were from intergenic regions. The function of this long non-coding RNA remains unknown but points clearly to an additional level of complexity in early embryo development. For all ten in vitro systems and the in vivo counterparts, three biological replicates were done. Technical duplicates for each of these 33 samples were analyzed to generate a dataset composed of 65 microarray hybridizations (one of the replicated array failed). During analysis, the in vivo derived blastocysts were used as reference. Following data pre-processing (that includes, background subtraction, intra-array normalization (Loess) and inter-array normalization (Quantile), all technical replication (replicated spots on the array and technically replicated arrays) were combined to generate a single value (duplicated spots were averaged and the median was drawn from duplicated arrays) that was used for dowstream significance analysis.

Dataset Information

Transcription profiling of Bos taurus immature oocytes and 8-cell embryos to assess the impact of the loss of sample representation caused by the use of global linear amplification (T7-IVT) during microarray hybridization sample preparation

Publications

Real-time monitoring of aRNA production during T7 amplification to prevent the loss of sample representation during microarray hybridization sample preparation.

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