RT-PCR and RT-qPCR have been the major standard assays for the relative quantification of alternatively spliced protein isoforms for a decade. With the advance of technology, droplet digital PCR (ddPCR) is becoming widely implemented for these types of analysis due to the high-precision, absolute quantification of nucleic acid target sequences with absolute quantities without the need for standard curves29,30. To gain more insights into the expression of Mcl-1L and Mcl-1S isoforms in neural progenitors, cDNA samples from hNPCs either exposed to EtOH (50 mM for 24 h) or control-untreated cells were processed by ddPCR utilizing a pair of differential primers and a set of probes for each isoform as FAM-probe detecting Mcl-1L isoform and HEX-probe detecting Mcl-1S isoform in a single reaction. As shown in Fig. 6a–d, untreated hNPCs mainly express Mcl-1L isoform with less than 10% Mcl-1S mRNA copies. On the other hand, consistent with RT-PCR and qRT-PCR analysis, EtOH caused a shift in Mcl-1L / Mcl-1S ratio by favoring Mcl-1S splicing over Mcl-1L (Fig. 6e). Interestingly, when the true copies of each isoform transcripts were determined and compared
