Differential microRNA expression analysis in blastocysts by whole mount in situ hybridization and reverse transcription quantitative polymerase chain reaction on laser capture microdissection samples

Goossens, Karen; Spiegelaere, Ward De; Stevens, Mieke; Burvenich, Christian; Spiegeleer, Bart De; Cornillie, Pieter; Zeveren, Alex Van; Soom, Ann Van; Peelman, Luc

doi:10.1016/j.ab.2012.01.005

Cited by 16 publications

(14 citation statements)

References 42 publications

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“…5E, G). The results of miR-155 expression analysis were in agreement with our previous findings [32]. There is no evidence of differential miR449b in ICM versus TE samples (P > 0.05; Fig.…”

Section: Mirna Target Gene Validationsupporting

confidence: 92%

“…Mature miRNA expression was quantified using the SYBR Green master mix (Exiqon) and specific LNA PCR primer sets for miR-218, miR-449b, and miR-155 (Exiqon). U6 snRNA, 5SrRNA, and RNU1A (Exiqon) were chosen as reference small RNAs for normalization, as described in [32].…”

Section: Mirna Expression Analysismentioning

confidence: 99%

“…Whole-mount in situ hybridization (WISH) was performed as described in [32] using the 3¢ and 5¢-digoxigenin (DIG)-labeled LNA-modified oligonucleotide probe (10 pM) for miR-218 (cat. No.…”

Section: Differential Mirna Expression Analysis In Bovine Blastocystsmentioning

confidence: 99%

“…Manual dissection was used to isolate the mural TE cells. The purity of the ICM and TE samples was determined by RT-qPCR for the ICM marker NANOG and the TE marker KRT18 [32,39]. The expression of miR-218, miR-449b, and the target genes NANOG and CDH2 was analyzed in 3 pools of 10 separated ICM and TE samples using SYBR green-based RT-qPCR as described before.…”

Section: Differential Mirna Expression Analysis In Bovine Blastocystsmentioning

confidence: 99%

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Regulatory microRNA Network Identification in Bovine Blastocyst Development

Goossens

Mestdagh²,

Lefever³

et al. 2013

Stem Cells and Development

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Mammalian blastocyst formation is characterized by two lineage segregations resulting in the formation of the trophectoderm, the hypoblast, and the epiblast cell lineages. Cell fate determination during these early lineage segregations is associated with changes in the expression of specific transcription factors. In addition to the transcription factor-based control, it has become clear that also microRNAs (miRNAs) play an important role in the post-transcriptional regulation of pluripotency and differentiation. To elucidate the role of miRNAs in early lineage segregation, we compared the miRNA expression in early bovine blastocysts with the more advanced stage of hatched blastocysts. Reverse transcription-quantitative PCR-based miRNA expression profiling revealed eight upregulated miRNAs (miR-127, miR-130a, miR-155, miR-196a, miR-203, miR-28, miR-29c, and miR376a) and four downregulated miRNAs (miR-135a, miR-218, miR-335, and miR-449b) in hatched blastocysts. Through an integrative analysis of matching miRNA and mRNA expression data, candidate miRNA-mRNA interaction pairs were prioritized for validation. Using an in vitro luciferase reporter assay, we confirmed a direct interaction between miR-218 and CDH2, miR-218 and NANOG, and miR-449b and NOTCH1. By interfering with the FGF signaling pathway, we found functional evidence that miR-218, mainly expressed in the inner cell mass, regulates the NANOG expression in the bovine blastocyst in response to FGF signaling. The results of this study expand our knowledge about the miRNA signature of the bovine blastocyst and of the interactions between miRNAs and cell fate regulating transcription factors.

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“…5E, G). The results of miR-155 expression analysis were in agreement with our previous findings [32]. There is no evidence of differential miR449b in ICM versus TE samples (P > 0.05; Fig.…”

Section: Mirna Target Gene Validationsupporting

confidence: 92%

Section: Mirna Expression Analysismentioning

confidence: 99%

Section: Differential Mirna Expression Analysis In Bovine Blastocystsmentioning

confidence: 99%

Section: Differential Mirna Expression Analysis In Bovine Blastocystsmentioning

confidence: 99%

See 2 more Smart Citations

Regulatory microRNA Network Identification in Bovine Blastocyst Development

Goossens

Mestdagh²,

Lefever³

et al. 2013

Stem Cells and Development

Self Cite

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“…some patterns may contain single cells while others may contain more than one cell on the same micropattern) and RT-qPCR using the cell lysate from the whole micropattern surface will result in averaging of the individual differences. To study specific cell population within a mixed population, LCM has been applied to frozen sections in genomic and proteomic studies in both clinical and research laboratories [327,331,[341][342][343] and to sections of chemically fixed tissues [344].…”

Section: Laser Cutting and Catapulting In Palm Microbeam From Carl Zeissmentioning

confidence: 99%

Technique and method development for intervertebral disc (IVD) research

Lee¹,

李芷恩²

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Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. The direct and indirect cost of managing back pain posts heavy socioeconomic burden to the society. Improved technologies and techniques together with well documented experiments will benefit the research field by saving effort in doing the optimization in every laboratory and avoiding experiment failure due to incomplete understanding of the procedures. These can accelerate scientific discovery, reduce the sacrifice of animals and enable a more effective use of funding.Nucleus pulposus (NP) is the central part of IVD. Differences in matrix compositions in human NP clinical samples demand different cell isolation protocols for optimal results but there is no clear guide about this to date. Sub-optimal protocols may result in low cell yield, limited reliability of results or even failure of experiments. We experimented different isolation protocols to study different parameters involved and suggested some rules for cell isolation in three main applications: RNA extraction for phenotyping, cell isolation for cell culture, and characterization by flow cytometry.In addition, instead of extracting RNA from isolated cells, extraction of RNA from tissues directly may avoid the change of RNA levels during the cell isolation process.However, extraction of RNA directly from human and large animal IVD tissue is technically challenging due to its tough nature, low cell-to-matrix ratio and high proteoglycan content. Thus we developed a method for RNA extraction from bovine disc tissues by integrating the use of cryosectioning, additional phase separation and high salt precipitation into conventional guanidinium thiocyanate based method. With this method, RNA could be extracted from the NP tissue directly but the concentration was low. A shift toward 270 nm was observed in its UV spectrum which was due to phenol contamination. This caused an overestimation of RNA concentration. Hence we developed a computational method based on UV spectra for correcting the In short, different methods related to IVD research were developed and optimized. With improved methods together with a better understanding of the underlying rationale, researchers can save time and cost in their experiments and reduce experiment failure rate. This will help to accelerate researches. New methods also enable studies which were not feasible in the past.(484 words)

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MicroRNA In Situ Hybridization on Whole-Mount Preimplantation Embryos

Goossens

Peelman

Soom

2014

Methods in Molecular Biology

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Whole-mount in situ hybridization (WISH) using antisense probes is widely used to visualize RNA sequences in embryos and to determine the precise site of expression in the different cells or tissues. The target sequence is hybridized with an antisense RNA probe, followed by visual or fluorescence detection to measure the site and level of expression. However, the detection of short RNA molecules is hampered by the reduced stringency of the probes for short transcripts. Here, we describe a procedure for WISH detection of short RNA molecules, like miRNAs, in mammalian preimplantation embryos using LNA-modified probes with high sensitivity and specificity.

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Differential microRNA expression analysis in blastocysts by whole mount in situ hybridization and reverse transcription quantitative polymerase chain reaction on laser capture microdissection samples

Cited by 16 publications

References 42 publications

Regulatory microRNA Network Identification in Bovine Blastocyst Development

Regulatory microRNA Network Identification in Bovine Blastocyst Development

Technique and method development for intervertebral disc (IVD) research

MicroRNA In Situ Hybridization on Whole-Mount Preimplantation Embryos

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