By now it should be evident that strand-specific RT-PCR should include a large number of controls, to account for the many ways by which an incorrect result can be generated. Most of these involve the reverse transcription reaction, since this is where most of the errors come from. The one essential control that should be run for every individual sample is:
- A primer-free cDNA reaction.
- Proof that self-primed cDNA is not amplified.
Other controls that should be included at least once for the experiment are:
- A template-free
- Absence of contamination of solutions/pippettes with target DNA.
reverse-transcriptase-free cDNA reaction.
- Absence of reverse-transcriptase activity during PCR.
exonuclease-I-free cDNA reaction.
- Proof that the exonuclease-I abolishes amplification of self-primed cDNA through early participation of the tagged-cDNA primer in PCR.
The PCR step for all these controls should also include tagged-cDNA primer, equivalent to the estimated carry-over from the cDNA reaction, in addition to the regular concentrations of tag primer and virus-specific primer necessary for the PCR. Through this, the controls will contain the complete primer composition of the experimental reactions, which (as explained above) is an essential condition for excluding possible false positives.
Whether or not false-positive results during strand-specific RT-PCR presents a major problem also depends on the question to be answered. If the surprising result is the lack of replication in a certain host, tissue or cell type then false-positive results are not a major issue. In contrast, if the absolute presence or absence of replication needs to be proven, then extreme care must be taken when conducting and interpreting the experiments.