Ilian M A, Gilmour R S, Bickerstaffe R
Molecular Biotechnology Group, AFSD, Lincoln University, Canterbury, New Zealand.
J Anim Sci. 1999 Apr;77(4):853-64. doi: 10.2527/1999.774853x.
We developed a ribonuclease protection assay (RPA) to quantify the mRNA mass of calpastatin and the catalytic subunits of calpains I and II in ovine and bovine tissues. The method is based on constructing standard curves using predetermined amounts of in vitro synthesized sense cRNA of the calpain system, hybridized with excess radiolabeled antisense counterprobes. This is possible because the vectors used for riboprobe preparation can be used to transcribe the sense cRNA required to generate the standard curves to quantify absolute calpain I, calpain II, and calpastatin mRNA levels. We used the RPA to study calpain I, calpain II, and calpastatin gene expression in ovine liver, heart, and skeletal muscle. The results revealed that calpain II gene expression was similar in the three tissues. However, the expression of calpain I and calpastatin genes indicates that each tissue has its unique pattern. We also analyzed the activity of calpain I, calpain II, and calpastatin by the conventional DEAE chromatographic method for comparison. The results indicated that the RPA is more repeatable than the DEAE method. Special features of the RPA as compared with DEAE chromatography are as follows; 1) the RPA is a reliable method for quantifying the expression of calpains in all tissues because it is not affected by the presence of inhibitors or activators, 2) the RPA method can be expanded to analyze the expression of the tissue-specific calpains simply by designing specific probes for them, and 3) the RPA requires a small amount of tissue. The described method will facilitate future studies on the gene expression of calpains and will contribute to determining their physiological functions.
我们开发了一种核糖核酸酶保护分析方法（RPA），用于定量绵羊和牛组织中钙蛋白酶抑制蛋白以及钙蛋白酶I和II催化亚基的mRNA含量。该方法基于使用预先确定量的体外合成的钙蛋白酶系统正义cRNA构建标准曲线，将其与过量的放射性标记反义互补探针杂交。这是可行的，因为用于制备核糖探针的载体可用于转录生成标准曲线所需的正义cRNA，以定量钙蛋白酶I、钙蛋白酶II和钙蛋白酶抑制蛋白的绝对mRNA水平。我们使用RPA研究了绵羊肝脏、心脏和骨骼肌中钙蛋白酶I、钙蛋白酶II和钙蛋白酶抑制蛋白的基因表达。结果显示，钙蛋白酶II基因在这三种组织中的表达相似。然而，钙蛋白酶I和钙蛋白酶抑制蛋白基因的表达表明每个组织都有其独特的模式。我们还通过传统的DEAE色谱法分析了钙蛋白酶I、钙蛋白酶II和钙蛋白酶抑制蛋白的活性以作比较。结果表明，RPA比DEAE方法更具可重复性。与DEAE色谱法相比，RPA的特点如下：1）RPA是一种可靠的方法，可用于定量所有组织中钙蛋白酶的表达，因为它不受抑制剂或激活剂存在的影响；2）RPA方法只需为组织特异性钙蛋白酶设计特定探针，就可扩展用于分析其表达；3）RPA所需的组织量较少。所描述的方法将有助于未来对钙蛋白酶基因表达的研究，并有助于确定它们的生理功能。
