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

Abstract

AbstractThe procedure illustrated in this paper represents a new method for transcriptome analysis by PCR (Polymerase Chain Reaction), which circumvents the need for elimination of potential DNA contamination. Compared to the existing methodologies, our method is more precise, simpler and more reproducible because it preserves the RNA’s integrity, does not require materials and/or reagents that are used for elimination of DNA and it also reduces the number of samples that should be set up as negative controls. This novel procedure involves the use of a specifically modified primer during reverse transcription step, which contains mismatched bases, thus producing cDNA molecules that differ from genomic DNA. By using the same modified primer in PCR amplification, only cDNA template is amplified since genomic DNA template is partially heterologous to the primer. In this way, amplification by PCR is unaffected by any potential DNA contamination since it is specific only for the cDNA template. Furthermore, it accurately reflects the initial RNA concentration of the sample, which is prone to changes due to various physical or enzymatic treatments commonly used by the current methodologies for DNA elimination. The method is particularly suitable for quantification of highly repetitive DNA transcripts, such as satellite DNA.