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mRNA quantitation by a simple and sensitive RNAse protection assay
M E Pape1, G W Melchior, K R Marotti
1Upjohn Company, Kalamazoo, Michigan 49001.
Abstract:
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.
Insights
This study introduces a precise RNAse protection assay using an internal standard to accurately measure messenger RNA (mRNA) levels in cells and tissues. The method quantifies specific apolipoprotein mRNA in cynomolgus monkey tissues.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Accurate quantification of mRNA levels is crucial for understanding gene expression.
- Existing methods may lack the precision required for detailed cellular and tissue analysis.
- A need exists for a reliable assay to measure specific mRNA populations.
Purpose of the Study:
- To develop and validate a highly precise RNAse protection assay for mRNA quantification.
- To establish a method for measuring apolipoprotein (apo)-A-I, apo-B, and apo-E mRNA levels in non-human primate tissues.
- To demonstrate the utility of an internal standard for enhancing assay accuracy.
Main Methods:
- Utilized RNAse protection assay with a synthesized internal RNA standard.
- Constructed RNA quantitation vectors (pRQV) for generating sense (internal standard) and antisense (probe) RNA strands.
- Performed solution hybridization, RNAse digestion, and gel electrophoresis to separate and quantify RNA fragments.
- Calculated authentic mRNA mass based on radioactivity ratios of protected fragments.
Main Results:
- The developed assay significantly increased the precision of mRNA level measurements.
- Successfully quantified apo-A-I, apo-B, and apo-E mRNA in cynomolgus monkey tissues.
- Demonstrated that distinct fragment sizes of internal standard and authentic mRNA-probe hybrids allow for accurate differentiation and quantification.
Conclusions:
- The RNAse protection assay with an internal standard provides a precise and versatile method for mRNA quantification.
- This technique is applicable to any mRNA for which corresponding cDNA is available.
- The assay is valuable for studying gene expression patterns in various biological contexts, including non-human primate models.
