Zusammenfassung
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. (C) 2009 Elsevier Inc. All rights
reserved.
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