REVERSE TRANSCRIPTION-QUANTITATIVE PCR (RT-QPCR) WITHOUT THE NEED FOR REMOVAL OF TEMPLATE DNA

One of the major problems in transcriptome analysis is inability to completely eliminate template DNA, which is indistinguishable from cDNA, thus resulting in false positive signals. We developed a novel method for transcriptome analysis by RT-qPCR (Reverse-Transcription quantitative Polymerase Chain Reaction), which circumvents the need for elimination of potential DNA contamination, therefore being more precise, simpler and more reproducible than the commonly used methods. The novel procedure involves the use of a modified specific primer during reverse transcription step, which contains mismatched bases, thus producing cDNA molecules not perfectly homologous to genomic DNA. By using the same modified primer in PCR amplification step, only cDNA template is amplified since genomic DNA template is not recognized by the primer. We determined the expression of Escherichia coli recA and sulA single-copy genes by RT-qPCR using either modified primers, or following the standard procedure. No recA and sulA sequence amplification was observed using our method unless cDNA was created by reverse transcription. The level of recA and sulA sequence amplification was unaffected by genomic DNA elimination from the sample. Conversely, the current method, which uses standard random/oligo-dT primers, showed a false positive signal even when reverse transcription step was skipped and the genomic DNA was (obviously incompletely) eliminated by DNase I treatment. Hence, our method of using a modified primer during cDNA synthesis produces a cDNA-specific PCR signal that is unaffected by genomic DNA and therefore quantifies gene expression much more accurately than the standard, commonly used method.