Quantification of Absolute Amounts of Cellular Messenger RNA by RNA-Excess Solution Hybridization

The quantification of mRNA in mammalian tissues or cell culture is an important part of analysis of gene expression and is required to fully understand many biological processes, such as cell replication, growth, and differentiation or the effects of hormonal, dietary, and genetical factors. The measurement of specific mRNAs relies on the hybridization of nucleic acids with labeled probes that have comple -mentary sequences. The most common analysis utilizes Northern or slot blotting. For Northern blot analysis, RNA is resolved by agarose gel electrophoresis, transferred to a nitrocellulose or nylon membrane and probed either with cDNA (1 ) or riboprobe (2 ,3 ). Slot blotting is performed without prior electrophoresis. Both methods are semiquantitative, unreliable for detecting small changes in the mes- sage levels, and provide only relative values of mRNA. By contrast, DNA-excess solution hybridization (4 ) can be used to quantify abso -lute levels of mRNA. In this method a cDNA fragment of a specific message is first subcloned into a M13 vector and single-stranded DNA prepared. The single-stranded DNA is used to synthesize a comple -mentary cDNA probe, which is then excised from the vector with appropriate restriction enzymes and isolated by denaturing polyacryla mide gel electrophoresis (4 ). A standard curve is generated by performing hybridization of the cDNA probe with the single-stranded M13 template. The RNA to be analyzed for the specific message is also hybridized and the absolute levels of RNA calculated by comparison of the counts obtained to the standard curve. Although DNA-excess hybridization is a great improvement over blotting methods, the disadvantage is that it requires lengthy preparation times and the quantification values are based on a standard curve derived from the DNA:DNA-hybridization of the cDNA probe to the single-stranded DNA template. Furthermore, we have found that single-stranded templates made from phagemids, such as pGem3Zf(+), are always contaminated with other DNAs, which affect the accuracy of quantification of the single-stranded DNA standard.