136 USE OF NANOFLUIDIC RT QUANTITATIVE POLYMERASE CHAIN REACTION TO STUDY VARIATION IN GENE EXPRESSION BETWEEN SINGLE BOVINE OOCYTE AND BLASTOCYST SAMPLES

Abstract

Most research into gene expression of oocyte and pre-implantation-stage embryos has been on pooled samples. Little work has been done at the single oocyte or early embryo levels. Our hypothesis was that a large amount of variation exists in relative transcript abundance between individual oocytes/embryos, which may ultimately be associated with inherent developmental potential. Because most studies to date have been on pooled samples, only very limited data have been presented that explain whether such variation exists, or if most oocytes/embryos within a pool are similar in gene expression. To generate preliminary information about the amount of variation in gene expression that exists between single oocytes and blastocyst-stage embryos, 4 single oocyte and 8 single blastocyst samples were compared with pooled oocyte samples of 10 and 20 and a pooled blastocyst sample of 10. Oocyte maturation and embryo production were performed according to established protocols (see Aston et al. 2010 Cell Reprog.). The 24 genes selected for use in this analysis consisted mostly of pluripotency genes, imprinted genes and housekeeping genes. To analyse such small samples we used nanofluidic BioMark^® quantitative PCR (qPCR) technology from Fluidigm (San Francisco, CA, USA). Complementary DNA was generated and preamplified by adding the samples listed above directly to a reverse-transcription/preamplification mix according to a prevalidated protocol from Fluidigm. Samples were run in duplicate on a single 48.48 microfluidic chip using intercalating dye qPCR technology. Each primer set was run in duplicate as well, resulting in 4 data points for each sample and primer set combination. Data were analysed using the relative standard curve method. We were able to produce preliminary data on how reproducible data generation is with this platform on single oocyte and blastocyst samples. Correlations between assay and sample replicates were high (R² = 0.998 and R² = 0.967, respectively). Among the oocyte samples, a fold change difference of 2 or more was found in 9 of the 24 primer sets. A similar analysis of the blastocyst samples yielded fold changes over 2 for 12 of the 24 primer sets. These preliminary data suggest that the use of nanofluidic qPCR technology may be useful for the study of gene expression variation among single oocyte and blastocyst samples.

Funding was provided to S. C. Isom through the Utah Agricultural Experiment Station (program project #UTA00151).