XBP1 splicing

Total RNA was extracted from mouse tissue using TRIzol reagent (Life Technologies), and reverse transcribed into cDNA using iScript cDNA Synthesis Kit (BIO-RAD). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed with iQ SYBR Green Supermix (BIO-RAD) using primer sets specific for target and reference genes (PrimerBank, MGH). The relative splicing level of Xbp1 was determined by normalizing the expression of spliced Xbp1(S) to unspliced Xbp1(U) mRNA. The relative transcription levels of all other genes were determined by normalizing the expression of target genes to the reference 18 s rRNA. Relative gene expression was calculated based on the reaction cycle threshold (Ct) values using Pfaff method⁵⁹. Primers: Xbp1s forward 5′-GAGTCCGCAGCAGGTG-3′ and reverse 5′-GTGTCAGAGTCCATGGGA-3′; Xbp1u forward 5′-GACTATGTGCACCTCTGCAG-3′ and reverse 5′-CTGGGAGTTCCTCCAGACTA-3′.