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 μl 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 rangin

SUBMITTER: Kimble KM 

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

REPOSITORIES: biostudies-literature