Detection and quantification of RNA are performed by Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR (RT-qPCR). These methods are widely used in molecular research and clinical diagnostics but are unable to discriminate between amplification of cDNA and genomic DNA. This could lead to the generation of false positive results and in the case of RT-qPCR, an incorrect estimation of the RNA quantity.
Several sources of genomic DNA contamination can occur in a RT-qPCR and one of the most common sources is from the cells used in RNA extraction. Often, PCRs are designed using intron-spanning primers, aiming to specifically amplify cDNA. However, intelligent primer design cannot guarantee accurate RNA quantification. Unless the intron is of sufficient size, contaminating gDNA could still compete for primers and probes and lead to false positives. Processed pseudogenes could also constitute a significant source of false positives. These pseudogenes do not contain introns, and are identical to the cDNA. Hence, it is prudent in RT-PCRs and RT-qPCRs to remove any contaminating DNA prior to amplification.
The most common method for removal of contaminating genomic DNA from RNA has been DNase I treatment. A new fast method that also is reliable and preserves all your RNA is here. No longer DNase I treatment – the Heat&Run gDNA removal kit is here.