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Comprehensive profiling of Small RNAs in human embryo-conditioned culture media by improved sequencing and quantitative PCR methods.

07:00 EST 13th February 2020 | BioPortfolio

Summary of "Comprehensive profiling of Small RNAs in human embryo-conditioned culture media by improved sequencing and quantitative PCR methods."

Embryo implantation depends on two primary factors: the quality of the embryo and endometrial receptivity. Small RNAs have been shown to be potent epigenetic regulators influencing cell proliferation, differentiation, and communication even in the context of early embryonic development. However, previous reports are limited to miRNAs and lack sensitivity. Here, we describe a platform for non-invasive small RNA biomarker discovery and validation from embryo-conditioned culture media (ECCM). We hypothesize that small non-coding RNAs (sncRNAs) are secreted by the embryo into the ECCM and test the limit of detection for profiling sncRNA by deep sequencing and quantitative PCR. In the first set of experiments, we evaluated sequencing sensitivity by comparing sncRNA profiles from pools of 10, 5, 3, and single ECCM drops. Next, we performed a similar test for TaqMan qPCR sensitivity by measuring select sncRNAs in 5, 3 and single drop ECCM pools. Finally, we compared the expression of an sncRNA panel by qPCR in single ECCM vs no-embryo control media . We report the first comprehensive sequencing of sncRNAs in ECCM with a sequencing sensitivity of 3 single embryo drops, capturing ~150 miRNAs and an abundance of tRNA-derived small RNAs (tsRNAs). We then profiled 15 sncRNAs by qPCR and determined that the assay maintains sensitivity in single ECCM drops. Finally, we found significant differences in these sncRNA expression between control and ECCM drops. Improving embryo selection is crucial for reducing time to pregnancy. Here we describe a sensitive technique for biomarker discovery by sequencing and qPCR validation in ECCM, demonstrating that the majority of sncRNAs are embryo derived. We also report an abundance of tsRNAs which suggests these sncRNAs may have functions in endometrial-maternal communication beyond the microRNAs which have been described previously. PGT-
A:
Preimplantation genetic testing for aneuploidies;
ECCM:
Embryo-conditioned culture media; sncRNAs: Small non-coding RNAs; miRNAs: microRNAs; EVs: Extracellular vesicles;
PCA:
Principal component analysis.

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Journal Details

This article was published in the following journal.

Name: Systems biology in reproductive medicine
ISSN: 1939-6376
Pages: 1-11

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Medical and Biotech [MESH] Definitions

Small double-stranded, non-protein coding RNAs, 21-25 nucleotides in length generated from single-stranded microRNA gene transcripts by the same RIBONUCLEASE III, Dicer, that produces small interfering RNAs (RNA, SMALL INTERFERING). They become part of the RNA-INDUCED SILENCING COMPLEX and repress the translation (TRANSLATION, GENETIC) of target RNA by binding to homologous 3'UTR region as an imperfect match. The small temporal RNAs (stRNAs), let-7 and lin-4, from C. elegans, are the first 2 miRNAs discovered, and are from a class of miRNAs involved in developmental timing.

Culture media containing biologically active components obtained from previously cultured cells or tissues that have released into the media substances affecting certain cell functions (e.g., growth, lysis).

The procedure of presenting the conditioned stimulus without REINFORCEMENT to an organism previously conditioned. It refers also to the diminution of a conditioned response resulting from this procedure.

Small RNAs found in the cytoplasm usually complexed with proteins in scRNPs (RIBONUCLEOPROTEINS, SMALL CYTOPLASMIC).

Small nuclear RNAs that are involved in the processing of pre-ribosomal RNA in the nucleolus. Box C/D containing snoRNAs (U14, U15, U16, U20, U21 and U24-U63) direct site-specific methylation of various ribose moieties. Box H/ACA containing snoRNAs (E2, E3, U19, U23, and U64-U72) direct the conversion of specific uridines to pseudouridine. Site-specific cleavages resulting in the mature ribosomal RNAs are directed by snoRNAs U3, U8, U14, U22 and the snoRNA components of RNase MRP and RNase P.

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