In order to analyze the ratio between human Mcl-1L and Mcl-1S isoforms, a quantitative RT-PCR was performed. hMcl-1L isoform coding region is composed of 3 exons: Exon 1, Exon 2 and Exon 3, while hMcl-1S isoform coding region is composed of 2 exons: Exon 1 and Exon 3. Two different probes, with fluorophore with different excitation/emission spectrum, were designed to anneal specifically the hMcl-1L or the hMcl-1S isoform, in correspondence of exons junction. hMcl-1L isoform probe anneals within the junction between Exon 2 and Exon 3, while hMcl-1S isoform probe anneals within the junction between Exon 1 and Exon 3. hMcl-1L isoform probe has FAM as fluorophore at 5′ and it is the following: 5′-CAAAGAGGCTGGGATGGGTTTGTG-3′. hMcl-1S isoform probe has HEX as fluorophore at 5′ and its sequence is: 5′-CGGCCTTCCAAGGATGGGTTTGTG-3′. The RT-qPCR was performed in LightCycler 480 (Roche Applied Science) using Luna® Universal Probe One-Step RT-qPCR Kit (New England Biolabs). Briefly, total RNA of treated cells was isolated using the TRIzol™ Reagent protocol. 100 ng of RNA was used as template. The RT-qPCR reaction mixtures contained: 1 × Luna Universal Probe
