and Mcl-1S isoforms. As shown in Fig. 5b, c, each probe showed differential and quantitative detection of both isoforms with FAM-probe detecting Mcl-1L isoform and HEX-probe detecting Mcl-1S isoform. Next, sensitivity of both probes was determined in dual probe reactions in the presence of both Mcl-1L and Mcl-1S standards. As shown in Fig. 5d, e, both Mcl-1L and Mcl-1S isoforms were specifically and selectively recognized by FAM and HEX-probes, respectively. Finally, quantitative amplification and differential detection of both isoforms were tested and confirmed in a single reaction in the presence of both Mcl-1L and Mcl-1S standards (Fig. 5f). Once the real-time qRT-PCR assay was established, the impact of EtOH exposure on alternative splicing of Mcl-1L and Mcl-1S isoforms was analyzed in different lineages of neuronal cells. hNSPs, hNPCs, immature neurons, and mature neurons were exposed to EtOH (50 mM) and total RNA extracts were processed by real-time qRT-PCR at 24 h post exposures. Consistent with semi-quantitative RT-PCR analysis (Fig. 4), EtOH exposure caused a significant shift in Mcl-1L and Mcl-1S ratio by favoring Mcl-1S alternative splicing over Mcl-1L in hNSPs, hNPCs, and immature neurons with no significant alteration in mature neurons in cultures. Moreover, dose response effects of EtOH on
