Rapid and simple comparison of messenger RNA levels using real-time PCR

Abstract

Real-time polymerase chain reaction (PCR) constitutes a significant improvement over traditional end-point PCR,as it allows the quantification of starting amounts of nucleic acid templates, in real-time. However,quantification requires validation through numerous internal controls and standard curves. We describe in this paper a simple protocol which uses real-time PCR to compare mRNA levels of a gene interest between different experiment conditions. Comparative real-time PCR can be a relatively low-cost method and does not require sequence-specific fluorescent reporters. Moreover, several genes from a set of experiments can be assessed in a single run. Thus,in addition to providing a comparative profile for the expression of a gene of interest, this method can also provide information regarding the relative abundance of different mRNA species. © 2006 by the author(s). This paper is Open Access and is published in Biological Procedures Online under license from the author(s).