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 One-Step Reaction Mix, 1 × Luna WarmStart RT Enzyme Mix, 400 nM of each primer, 400 nM of each probe, template RNA and nuclease-free water to a final volume of 20 μL. The RT-qPCR protocol was: reverse transcription at 55 °C for 10 min, initial denaturation at 95 °C for 1 min, then 45 cycles of denaturation at 95 °C for 15 s and extensions at 60 °C for 1 min with single acquisition. The primers used were designed to anneal both isoforms within Exon 1 (Forward) and Exon 3 (Reverse) and are the following: H1-hMcl-1-F: 5′-GGACACAAAGCCAATGGGCAGGT-3′ and H3-hMcl-1-R: 5′-GCAAAAGCCAGCAGCACATTCCTGA-3′. pcDNA3.1-Mcl-1L and pcDNA3.1-Mcl-1S were used as standards and controls for specificity of the probes.
