Real time RT PCR
Two-step and one-step RT-PCR
Choice of RT primers
Conditions for two-step RT-PCR
With high GC content, the tight association of RNA:DNA hybrids can interfere with primer binding during PCR and prevent DNA polymerases from progressing (see figure Effect of high GC content on RT-PCR: PCR effects). RNase H removes RNA in RNA:DNA hybrids to allow primer binding and second-strand DNA synthesis. RNase H digestion has been previously shown to improve RT-PCR yield and to be required for amplification of some sequences, even as short as 157 bp (7).
Conditions for one-step RT-PCR
To detect amplification of contaminating DNA, RT-PCR primers should be designed to flank a region that contains at least one intron. Products amplified from cDNA (no introns) will be smaller than those amplified from genomic DNA (containing introns). Size difference in products is used to detect the presence of contaminating DNA.
If only the mRNA sequence is known, choose primer annealing sites that are at least 300–400 bp apart. It is likely that fragments of this size from eukaryotic DNA contain splice junctions. As explained in the previous point, such primers may be used to detect DNA contamination.
In summary, the following factors should be considered when designing primers for RT-PCR:
Annealing temperature can affect RT-PCR efficiency and sensitivity.
High primer concentrations can cause mispriming and primer–dimer formation.
A stringent hot start is essential for optimal RT-PCR performance.
Primer design in RT-PCR allows differentiation of signals from RNA and contaminating DNA. For best results, DNA-free RNA should be used in order to avoid competition of DNA in RT-PCR.