RNA Template-Specific Polymerase Chain Reaction (RS-PCR): A Modification of RNA-PCR that Dramatically Reduces the Frequency of False Positives

Reverse transcription of RNA followed by the polymerase chain reaction (RT-PCR or RNA-PCR) is an extraordinarily sensitive method to detect as few as 1–100 copies of a specific RNA (1 -3 ). However, we and others have found that false positives caused by contamination with minute quantities of DNA (i.e., cDNAs, plasmid DNAs, genomic DNA, or PCR carryover) is a major shortcoming of the method even when meticulous laboratory technique is employed (4 -6 ). RNA template-specific PCR (RS-PCR) is a modification of conventional RNA-PCR in which RNA is reverse-transcribed with a primer that contains at its 5′ end a unique nucleotide sequence that may then be exploited in the PCR to amplify preferentially the RNA-derived sequence. RS-PCR retains full sensitivity, but reduces dramatically the frequency of false positives (7 -9 ).