I plan to measure the effects of Tenofovir a Nucleotide analog reverse-transcriptase inhibitors useing qrt-pcr with HIV-RT as the rt enzyme. Tenofovir causes early termination of the reverse transcriptase by binding to reads preventing the continuation of the chain process rather than stopping the action all together. How will qrt-pcr quantify the low molecular weight short incomplete fragments? I think since the areas complementary to the forward and reverse primers will rarely be on the same continuous double stranded dna expression will be decreased uniformly. Is this correct? Will I need to use absolute qrt-pcr since the changes will be the same for all genes?
You have to design your primers properly. Usually, in real-time PCR, you don't choose a very long product. Ideal product size is 150-300.
Next, see what your NRTI is analogous to. For e.g. if I am using AZT (Azathymidie), I would place my reverse primer at or after the last
There are alternate techniques as well. You can use primer extension (radiolabeled) and then quantify different products by scanning the photograph. However, this technique won't be as quantitative as real-time PCR.