2.5. XBP1 mRNA Splicing Assay

Total cellular RNA was purified using Trizol reagent (Thermo Fisher Scientific, Hampton, NH, USA) according to the manufacturer’s instructions. RT-PCR was performed by using PrimeScript™ One Step RT-PCR Kit Ver.2 (TaKaRa Bio Inc., Otsu, Japan) [39,40,41]. The XBP1 gene was amplified by PCR with XBP1-F: 5′-CTGAAAAACAGAGTAGCAGCTCAGA-3′ and XBP1-R: 5′-TCAGTTCATTAATGGCTTCCAGC-3′. The spliced (XBP1s) and unsliced (XBP1u) forms were analyzed by digesting the RT-PCR products with the restriction enzyme Pst I (FD0615) (Thermo Fisher Scientific, Hampton, NH, USA). Followed by purification of digested PCR products with 1.5% agarose gel electrophoresis and analysis by using the gel imaging system (Federal bioproducts Inc., Manufacturer, UK) [28,42,43].