Quantifying cytosolic messenger RNA concentrations in Escherichia coli using real-time polymerase chain reaction for a systems biology approach.

Current messenger RNA (mRNA) quantification methods are sophisticated tools for the analysis of gene regulation. However, these methods are not suitable for more complex quantitative approaches such as the mathematical modeling of the in vivo regulation of transcription where dynamic cytosolic mRNA concentrations need to be taken into consideration. In the current study, the "standard curve method" for quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR) was extended by including an internal RNA standard. This standard enables transcript losses that occur during the process, as well as variations resulting from nonquantitative processes, to be accounted for. The use of an internal standard yielded transcript concentration estimates that were on average seven times higher than those in cases where an internal standard is omitted. Choosing the cra modulon in Escherichia coli as an example, the method applied shows that the regulation of the Cra protein, as well as the growth rate-dependent regulation, need to be taken into consideration. The new method, which enables the determination of cytosolic mRNA concentrations, allows the quantitative representation of transcriptional dynamics. This is an important aspect of the analysis of the complex interactions of metabolism and regulation and in the application of mathematical modeling for systems biology.