mRNA quantitation by a simple and sensitive RNAse protection assay.
An RNAse protection assay is described that increases substantially the degree of precision with which one can measure the mRNA levels in cells and tissues through the use of the internal standard. The assay can be used to measure any mRNA for which the corresponding cDNA is available. We describe here the use of the assay to measure the apolipoprotein (apo)-A-I, apo-B, and apo-E mRNA levels in tissues from the cynomolgus monkey. cDNA fragments derived from each mRNA were subcloned into pGEM-9Zf(-), a vector containing a polylinker that is flanked by the SP6 and T7 RNA polymerase promoters. That series of plasmids, called RNA quantitation vectors (pRQV-AI, B, or E), permitted the synthesis of a sense RNA strand and an antisense RNA strand for the gene of interest. The sense stand was used as the internal standard and added to the RNA to be analyzed just prior to initiation of the assay. The radiolabeled antisense strand served as the probe. By including some nucleotides derived from the vector, we were able to design both the internal standard and the probe such that, after solution hybridization and RNAse digestion, the size of the protected internal standard-probe fragments was different from that of the authentic mRNA-probe fragments. Those fragments were then separated by gel electrophoresis, and the radioactivity in the authentic mRNA band was compared to that in the internal standard band. The mass of the authentic mRNA could then be calculated from the ratio of the radioactivity in each band and the mass of the internal standard.
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Upjohn Company, Kalamazoo, Michigan 49001.
Melchior
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Marotti
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D000818:Animals; D016632:Apolipoprotein A-I / Q000032:analysis / Q000235:genetics; D001055:Apolipoproteins B / Q000032:analysis / Q000235:genetics; D001057:Apolipoproteins E / Q000032:analysis / Q000235:genetics; D003001:Cloning, Molecular; D005821:Genetic Techniques; D005822:Genetic Vectors; D008252:Macaca fascicularis; D009928:Organ Specificity / Q000235:genetics; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis*; D015183:Restriction Mapping; D012260:Ribonucleases / Q000378:metabolism*
D016632:Apolipoprotein A-I; D001055:Apolipoproteins B; D001057:Apolipoproteins E; D015347:RNA Probes; D012333:RNA, Messenger; D012260:Ribonucleases