National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Chungcheongbuk-do, Republic of Korea.
Futuristic Animal Resource & Research Center, Korea Research Institute of Bioscience and Biotechnology, Chungcheongbuk-do, Republic of Korea.
PLoS One. 2019 Jul 22;14(7):e0219978. doi: 10.1371/journal.pone.0219978. eCollection 2019.
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.
X 盒结合蛋白 1（XBP1）mRNA 处理在未折叠蛋白反应（UPR）中起着至关重要的作用，该反应是对内质网（ER）应激的反应而被激活的。当 UPR 转换的 XBP1mRNA 从未剪接（u）XBP1（非活性）异构体到剪接（s）XBP1（活性）异构体的剪接时，肌醇需求酶 1α（IRE1α）从 uXBP1mRNA 中去除 26 个核苷酸的内含子。最近的研究报告通过 densitometric 分析 PCR 带相对于使用管家基因进行标准化的 sXBP1 对 uXBP1 的增加水平来评估 ER 应激，该分析通过 densitometric 分析 PCR 带相对于 sXBP1 对 uXBP1 的增加水平来检查 sXBP1 与 uXBP1mRNA（s/uXBP1 比）的比值。然而，这种测量是通过凝胶电泳可视化的，这使得很难定量两个 XBP1 带之间的差异，并使数据解释复杂化。此外，大多数常用的管家基因在不同的实验条件下显示出 s/uXBP1 比值的不可接受的高可变表达模式，例如发育的不同阶段和不同的细胞类型，限制了它们作为内部对照的使用。为了更定量地确定 XBP1 剪接活性，我们通过使用特定的引物对通过实时 PCR 测量总 XBP1（tXBP1：s/uXBP1 的共同区域）和 sXBP1 的表达水平。我们还设计了通用的实时 PCR 引物对，能够扩增真核生物（包括人类、猴子、牛、猪和小鼠）中每个 u/s/tXBP1mRNA 的一部分，这些部分高度保守。因此，我们提供了一种更方便、更易于接近的定量实时 PCR 方法，可用于各种研究领域评估 ER 应激。
