Real-time PCR quantification of spliced X-box binding protein 1 (XBP1) using a universal primer method

Abstract

X-box binding protein 1 (XBP1) mRNA processing plays a crucial role in the unfolded protein response (UPR), which is activated in response to endoplasmic reticulum (ER) stress. Upon accumulation of the UPR-converted XBP1 mRNA splicing from an unspliced (u) XBP1 (inactive) isoform to the spliced (s) XBP1 (active) isoform, inositol-requiring enzyme 1 α (IRE1α) removes a 26-nucleotide intron from uXBP1 mRNA. Recent studies have reported the assessment of ER stress by examining the ratio of sXBP1 to uXBP1 mRNA (s/uXBP1 ratio) via densitometric analysis of PCR bands relative to increased levels of sXBP1 to uXBP1 using a housekeeping gene for normalization. However, this measurement is visualized by gel electrophoresis, making it very difficult to quantify differences between the two XBP1 bands and complicating data interpretation. Moreover, most commonly used housekeeping genes display an unacceptably high variable expression pattern of the s/uXBP1 ratio under different experimental conditions, such as various phases of development and different cell types, limiting their use as internal controls. For a more quantitative determination of XBP1 splicing activity, we measured the expression levels of total XBP1 (tXBP1: common region of s/uXBP1) and sXBP1 via real-time PCR using specific primer sets. We also designed universal real-time PCR primer sets capable of amplifying a portion of each u/s/tXBP1 mRNA that is highly conserved in eukaryotes, including humans, monkeys, cows, pigs, and mice. Therefore, we provide a more convenient and easily approachable quantitative real-time PCR method that can be used in various research fields to assess ER stress.

Endoplasmic reticulum associated degradation is required for maintaining endoplasmic reticulum homeostasis and viability of mature Schwann cells in adults

“…Reverse transcription was performed using the iScript™ cDNA Synthesis Kit (Bio‐Rad Laboratories, Hercules, CA). Real‐time PCR for spliced XBP1 mRNA (XBP1s) was performed with LightCycler® 480 SYBR Green I Master (Roche Diagnostics Corporation, Indianapolis, IN) on the LightCycler 480 System (Roche Diagnostics Corporation) as described in a previous paper (Yoon et al, 2019).…”

Unraveling the role of the enigmatic MatK maturase in chloroplast group IIA intron excision

“…For this reason, qPCR was chosen as the method to quantitatively ascertain splicing efficiency. qPCR has been shown previously to provide accurate quantitative detection of spliced products, even more so than densitometry of products resolved by gel electrophoresis (Yoon et al, 2019). qPCRs were performed using SsoAdvanced™ Universal SYBR ® Green Supermix (Bio‐Rad) on the Bio‐Rad CFX 96 Touch™ Real‐time PCR Detection System with at least two technical replicates for each sample and a melting curve used to assess specificity of product formation in the reaction.…”

Establishment and Characterization of Immortalized Miniature Pig Pancreatic Cell Lines Expressing Oncogenic K-RasG12D

“…Co, Chungcheongbuk-do, Republic of Korea), and all efforts were made to minimize animal suffering by institutional veterinary experts, in accordance with National Institutes of Health (NIH) guidelines. A 2-week-old KSP miniature pig [ 45 ] was provided by the Futuristic Animal Resource & Research Center in KRIBB, Korea. Primary pancreatic cells (PCs) were prepared from the miniature pig pancreas as preciously described [ 46 ] and maintained in RPMI 1640 medium (#11875119, GIBCO, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, #16000-044, GIBCO), 100 U/mL penicillin and 100 µg/mL streptomycin (#15140122, GIBCO).…”