MicroRNA In Situ Hybridization on Whole-Mount Preimplantation Embryos

Goossens, Karen; Peelman, Luc; Soom, Ann Van

doi:10.1007/978-1-4939-1459-3_2

Cited by 2 publications

(1 citation statement)

References 17 publications

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“…At this time, expanded blastocysts were selected and fixed in 4% paraformaldehyde (PFA) (Sigma) for 1 h for later in situ analysis. Blastocysts were prepared according to Goossens et al (2014) with several modifications. Briefly, fixed blastocysts were dehydrated in methanol at −20 • C overnight followed by rehydration in a methanol/PBST series and a 30-s digestion with proteinase K (Sigma).…”

Section: Blastocyst In Situ Hybridization and Confocal Analysismentioning

confidence: 99%

Murine Blastocysts Release Mature MicroRNAs Into Culture Media That Reflect Developmental Status

et al. 2021

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Extracellular microRNA (miRNA) sequences derived from the pre-implantation embryo have attracted interest for their possible contributions to the ongoing embryonic–uterine milieu, as well as their potential for use as accessible biomarkers indicative of embryonic health. Spent culture media microdroplets used to culture late-stage E4.0 murine blastocysts were screened for 641 mature miRNA sequences using a reverse transcription–quantitative polymerase chain reaction–based array. We report here 39 miRNAs exclusively detected in the conditioned media, including the implantation-relevant miR-126a-3p, miR-101a, miR-143, and miR-320, in addition to members of the highly expressed embryonic miR-125 and miR-290 families. Based on these results, an miRNA panel was assembled comprising five members of the miR-290 family (miR-291-295) and five conserved sequences with significance to the embryonic secretome (miR-20a, miR-30c, miR-142-3p, miR-191, and miR-320). Panel profiling of developing embryo cohort lysates and accompanying conditioned media microdroplets revealed extensive similarities in relative quantities of miRNAs and, as a biomarker proof of concept, enabled distinction between media conditioned with differently staged embryos (zygote, 4-cell, and blastocyst). When used to assess media conditioned with embryos of varying degrees of degeneration, the panel revealed increases in all extracellular panel sequences, suggesting cell death is an influential and identifiable factor detectable by this assessment. In situ hybridization of three panel sequences (miR-30c, miR-294, and miR-295) in late-stage blastocysts revealed primarily inner cell mass expression with a significant presence of miR-294 throughout the blastocyst cavity. Furthermore, extracellular miR-290 sequences responded significantly to high centrifugal force, suggesting a substantial fraction of these sequences may exist within a vesicle such as an exosome, microvesicle, or apoptotic bleb. Together, these results support the use of extracellular miRNA to assess embryonic health and enable development of a non-invasive viability diagnostic tool for clinical use.

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Section: Blastocyst In Situ Hybridization and Confocal Analysismentioning

confidence: 99%

Murine Blastocysts Release Mature MicroRNAs Into Culture Media That Reflect Developmental Status

et al. 2021

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Profiling bovine blastocyst microRNAs using deep sequencing

Pasquariello

Fernández-Fuertes

Strozzi

et al. 2017

Reprod. Fertil. Dev.

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MicroRNAs (miRNAs) are known to control several reproductive functions, including oocyte maturation, implantation and early embryonic development. Recent advances in deep sequencing have allowed the analysis of all miRNAs of a sample. However, when working with embryos, due to the low RNA content, miRNA profiling is challenging because of the relatively large amount of total RNA required for library preparation protocols. In the present study we compared three different procedures for RNA extraction and prepared libraries using pools of 30 bovine blastocysts. In total, 14 of the 15 most abundantly expressed miRNAs were common to all three procedures. Furthermore, using miRDeep discovery and annotation software (Max Delbrück Center), we identified 1363 miRNA sequences, of which bta-miR-10b and bta-miR-378 were the most abundant. Most of the 179 genes identified as experimentally validated (86.6%) or predicted targets (13.4%) were associated with cancer canonical pathways. We conclude that reliable analysis of bovine blastocyst miRNAs can be achieved using the procedures described herein. The repeatability of the results across different procedures and independent replicates, as well as their consistency with results obtained in other species, support the biological relevance of these miRNAs and of the gene pathways they modulate in early embryogenesis.

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MicroRNA In Situ Hybridization on Whole-Mount Preimplantation Embryos

Cited by 2 publications

References 17 publications

Murine Blastocysts Release Mature MicroRNAs Into Culture Media That Reflect Developmental Status

Murine Blastocysts Release Mature MicroRNAs Into Culture Media That Reflect Developmental Status

Profiling bovine blastocyst microRNAs using deep sequencing

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