Competitive RT-PCR to Quantify Small Amounts of mRNA

Abstract

Monitoring cytokine responses to various stimuli or determining the expression pattern of cytokine mRNAs requires sensitive technologies for cytokine mRNA quantitation where there is a limited quantity of material, for example, when working with biopsies. Owing to their amplifying effect, reverse transcriptase-polymerase chain reacttion (RT-PCR)-based methods permit the analysis of minimal starting quantities of nucleic acids. Because of the exponential nature of PCR, introduction of a competitive internal standard (competitor) has proven to be of great advantage for quantitation. Competitive RT-PCR is capable of ruling out tube-to-tube and sample-to-sample variation, because the competitor and the target of interest are amplified in the same reaction tube, compete for the same enzyme and nucleotides, and, thus, are subjected to identical amplification conditions (1,2). This is achieved by the special construction of the competitor, which bears the same primer-binding regions as the target of interest, but the sequence in between is modified in such a way that amplification products derived from the competitor and the target can be differentiated.