Reverse transcription-quantitative PCR (RT-qPCR) without the need for removal of template DNA

The qualitative and/or quantitative analysis of transcripts by PCR amplification is widely used to detect and quantify many different types of transcripts such as messenger RNA, ribosomal RNA, non-coding RNA etc. The most common applications include gene expression analysis and precise identification of a particular microorganism. For the purified RNA to be quantified, it is subjected to reverse transcription (RT), by which the RNA molecules are converted into cDNA (complementary DNA) by the reverse transcriptase enzyme. The main problem of most currently used protocols lies in the often-present DNA contamination, which is impossible to chemically differentiate from the cDNA by the polymerase enzyme during PCR amplification, thus causing false positive results. To overcome this limitation, all current protocols include a couple of DNA elimination steps, both during the purification of RNA as well as in subsequent RT step. The DNA is eliminated either with the aid of specific mechanical filters (silica-based columns) or through enzymatic digestion by a specific enzyme, such as DNaseI (Deoxyribonuclease I). However, these treatments are not 100% effective, often leaving DNA contamination. Furthermore, any procedure implemented to reduce the concentration of DNA in the sample certainly causes the reduction of the RNA concentration as well, which is unstable and easily degradable molecule. Here we report a novel method for transcriptome analysis by PCR, wherein cDNA and template DNA are differentiated and thus contamination by the latter is excluded, hence producing more precise, reliable and reproducible results.