Quantifying BRCA1 and BRCA2 mRNA Isoform Expression Levels in Single Cells

and spliceogenic variants are often associated with an elevated risk of breast and ovarian cancers. Analyses of and splicing patterns have traditionally used technologies that sample a population of cells but do not account for the variation that may be present between individual cells. This novel proof of concept study utilises RNA in situ hybridisation to measure the absolute expression of and mRNA splicing events in single lymphoblastoid cells containing known spliceogenic variants ( c.671-2 A>G or c.7988 A>T). We observed a large proportion of cells (>42%) in each sample that did not express mRNA for the targeted gene. Increased levels (average mRNA molecules per cell) of ∆17_18 were observed in the cells containing the known spliceogenic variant c.7988 A>T, but cells containing c.671-2 A>G were not found to express significantly increased levels of ∆11, as had been shown previously. Instead, we show for each variant carrier sample that a higher proportion of cells expressed the targeted splicing event compared to control cells. These results indicate that / mRNA is expressed stochastically, suggesting that previously reported results using RT-PCR may have been influenced by the number of cells with / mRNA expression and may not represent an elevation of constitutive mRNA expression. Detection of mRNA expression in single cells allows for a more comprehensive understanding of how spliceogenic variants influence the expression of mRNA isoforms. However, further research is required to assess the utility of this technology to measure the expression of predicted spliceogenic and variants in a diagnostic setting.