from Vijay J. Gadkar and Martin Filion writing in Quantitative Real-time PCR in Applied Microbiology:
The first step towards analysing microbial gene expression requires a quantitative extraction of RNA. This step has proven to be highly problematic for environmental matrices, due to compounded inefficiencies in individual steps which include, but not limited to, incomplete cell lysis, RNA degradation by ubiquitous RNases, co-extraction of inhibitors and their interaction with the enzymes used. One straightforward approach to quantify such losses and apply the necessary correction is to include an internal amplification control (IAC), so as to make the final gene expression meaningful and reproducible. An IAC is essentially a non-target DNA/RNA sequence co-amplified, preferably in the same reaction tube, under the same reaction conditions. While attempts to develop IAC’s have met with some success for experimental systems which are highly controlled, developing such controls have proven to be highly problematic for certain experimental set-ups, for example complex environmental matrices. The main difficulty in these cases has been in our inability to identify an inert IAC which is able to (a) withstand the harsh nucleic acid extraction procedures usually employed for environmental matrices, and if such a sequence is indeed developed/ identified (b) designing a primer/probe combination which would not cross-react with other non-target (nucleic acids) components of the matrices. While few potential IAC based solutions have been proposed, for example the Biotrove OpenArray platform, high costs and proprietary issues of some IAC sequences have served as a deterrent for researchers who are seriously interested in rigorously implementing this external normalization strategy.
Further reading: Quantitative Real-time PCR in Applied Microbiology