Quantification of ovine and bovine calpain I, calpain II, and calpastatin mRNA by ribonuclease protection assay.
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.
D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002154:Calpain / Q000235:genetics*; D020134:Catalytic Domain / Q000235:genetics; D002417:Cattle / Q000235:genetics*; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics*; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D015347:RNA Probes / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*; D012756:Sheep / Q000235:genetics*