The RT-PCR assay provided semi-quantitative analysis of long (L) and short (S) isoforms of Mcl-1 pre-mRNA. In order to quantify the alternative splicing of both isoforms in control and EtOH exposed neuronal cells, we developed a unique real-time qRT-PCR based assay by utilizing probes specifically designed at exon junctions of Mcl-1 isoforms and two fluorophores with different excitation/emission spectrum. Mcl-1L probe was designed for the exons 2/3 junction with a FAM fluorophore and Mcl-1S probe was designed for exons 1/3 junction with a HEX fluorophore (Fig. 5a). Multiplexing approach was taken to amplify, distinguish, and quantify Mcl-1L and Mcl-1S isoforms in a single reaction. Mcl-1L and Mcl-1S isoforms were cloned into a mammalian expression vector and utilized as standards. The specificity of each probe was first established in a single probe reaction in the presence of both standards for Mcl-1L 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
