Tight gene co-expression in BCB positive cattle oocytes and their surrounding cumulus cells.

BACKGROUND: Cytoplasmic and nuclear maturation of oocytes, as well as interaction with the surrounding cumulus cells, are important features relevant to the acquisition of developmental competence. METHODS: Here, we utilized Brilliant cresyl blue (BCB) to distinguish cattle oocytes with low activity of the enzyme Glucose-6-Phosphate Dehydrogenase, and thus separated fully grown (BCB positive) oocytes from those in the growing phase (BCB negative). We then analyzed the developmental potential of these oocytes, mitochondrial DNA (mtDNA) copy number in single oocytes, and investigated the transcriptome of single oocytes and their surrounding cumulus cells of BCB positive versus BCB negative oocytes. RESULTS: The BCB positive oocytes were twice as likely to produce a blastocyst in vitro compared to BCB- oocytes (P < 0.01). We determined that BCB negative oocytes have 1.3-fold more mtDNA copies than BCB positive oocytes (P = 0.004). There was no differential transcript abundance of genes expressed in oocytes, however, 172 genes were identified in cumulus cells with differential transcript abundance (FDR < 0.05) based on the BCB staining of their oocyte. Co-expression analysis between oocytes and their surrounding cumulus cells revealed a subset of genes whose co-expression in BCB positive oocytes (n = 75) and their surrounding cumulus cells (n = 108) compose a unique profile of the cumulus-oocyte complex. CONCLUSIONS: If oocytes transition from BCB negative to BCB positive, there is a greater likelihood of producing a blastocyst, and a reduction of mtDNA copies, but there is no systematic variation of transcript abundance. Cumulus cells present changes in transcript abundance, which reflects in a dynamic co-expression between the oocyte and cumulus cells.

Delayed processing of blood samples impairs the accuracy of mRNA-based biomarkers.

The transcriptome of peripheral white blood cells (PWBCs) are indicators of an organism's physiological state, thus making them a prime biological sample for mRNA-based biomarker discovery. Here, we designed an experiment to evaluate the impact of delayed processing of whole blood samples on gene transcript abundance in PWBCs. We hypothesized that storing blood samples for 24 h at 4 °C would cause RNA degradation resulting in altered transcriptome profiles. There were no statistical differences in RNA quality parameters among samples processed after one, three, six, or eight hours post collection. Additionally, no significant differences were noted in RNA quality parameters or gene transcript abundance between samples collected from the jugular and coccygeal veins. However, samples processed after 24 h of storage had a lower RNA integrity number value (P = 0.03) in comparison to those processed after one hour of storage. Using RNA-sequencing, we identified four and 515 genes with differential transcript abundance in samples processed after storage for eight and 24 h, respectively, relative to samples processed after one hour. Sequencing coverage of transcripts was similar between samples from the 24-h and one-hour groups, thus showing no indication of RNA degradation. This alteration in transcriptome profiles can impair the accuracy of mRNA-based biomarkers, therefore, blood samples collected for mRNA-based biomarker discovery should be refrigerated immediately and processed within six hours post-sampling.