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

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 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.