Dataset Information

Extraction of total RNA from single-oocytes and single-cell mRNA sequencing of swine oocytes.

ABSTRACT:

Objective

Analyses of single oocytes are essential for a fine dissection of molecular features governing developmental competence. We adapted the phenol-chloroform procedure for the purification of total RNA from single oocytes.

Results

Key modifications include the use of Phasemaker™ tubes, a second chloroform wash of the aqueous phase, and the precipitation of the RNA with glyclogen in a 200 ?l micro-centrifuge tube. Assessment of the RNA profile from single oocytes showed distinct peaks for 18S and 28S ribosomal subunits. This approach permitted the extraction of small RNAs from single oocytes, which was evident by the presence of 5S and 5.8S rRNAs and tRNAs around 122-123 nucleotides long. The amplification of polyadenylated RNA resulted in detectable DNA products ranging from ~ 500 to ~ 5000 nucleotides. We used the amplified DNA as template for single-cell mRNA-sequencing of five swine oocytes and quantified the expression levels of 9587 genes with complete coverage of transcripts over 10,000 nucleotides in length. The coverage was similar in all oocytes sequenced, demonstrating consistent high RNA quality across samples. We isolated total RNA from single oocytes and demonstrated that the quality was appropriate for single-cell mRNA-sequencing.

SUBMITTER: Kimble KM

PROVIDER: S-EPMC5828076 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Publications

Extraction of total RNA from single-oocytes and single-cell mRNA sequencing of swine oocytes.

Kimble Katelyn M KM Dickinson Sarah E SE Biase Fernando H FH

BMC research notes 20180227 1

<h4>Objective</h4>Analyses of single oocytes are essential for a fine dissection of molecular features governing developmental competence. We adapted the phenol-chloroform procedure for the purification of total RNA from single oocytes.<h4>Results</h4>Key modifications include the use of Phasemaker™ tubes, a second chloroform wash of the aqueous phase, and the precipitation of the RNA with glyclogen in a 200 μl micro-centrifuge tube. Assessment of the RNA profile from single oocytes showed disti ...[more]

PMID: 29482623

Similar Datasets

Project description:BackgroundAgar-like zona pellucida (ZP) is the most common type of abnormal ZP, and is one of the causes of low fertility or infertility. However, the molecular mechanism of agar-like ZP is unclear. Single-cell RNA-sequencing (scRNA-seq) analysis was used to assess the cellular and molecular landscape of oocytes with agar-like ZP.MethodsHuman metaphase I (MI) oocytes were collected from four patients with agar-like ZP and four healthy donors. Total RNA was isolated, cDNA was synthesized, and libraries were generated and subsequently sequenced on a HiSeq 2500 instrument. The scRNA-seq data were analyzed with R software.ResultsWe identified 1320 genes that were differentially expressed between agar-like ZP oocytes and healthy donor oocytes. Gene Ontology term enrichment results showed that the genes downregulated in agar-like ZP oocytes were significantly related to extracellular matrix organization, while the genes upregulated in agar-like ZP oocytes were significantly related to the regulation of response to DNA damage stimulus. The Kyoto Encyclopedia of Genes and Genomes enrichment results showed that genes were enriched in the ECM-receptor interaction pathway and focal adhesion pathway. Other signaling pathways important in oocyte development were also enriched, such as PI3K-Akt. Differential expression analysis identified UBC, TLR4, RELA, ANXA5, CAV1, KPNA2, CCNA2, ACTA2, FYN and ITGB3 as genetic markers of oocytes with agar-like ZP.ConclusionsOur findings suggest that agar-like ZP oocytes exhibit significant downregulation of genes involved in the ECM-receptor interaction signaling pathway and focal adhesion pathway, which could lead to aberrant ZP formation, while the upregulated genes were significantly related to regulation of the response to DNA damage stimulus. Agar-like ZP formation may interfere with the normal exchange of signals between oocytes and perivitelline granulosa cells, thereby preventing cumulus cells from participating in oocyte DNA damage repair and causing MI arrest.

Dataset Information

Extraction of total RNA from single-oocytes and single-cell mRNA sequencing of swine oocytes.

Objective

Results

Publications

Extraction of total RNA from single-oocytes and single-cell mRNA sequencing of swine oocytes.

Similar Datasets

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