Quantitative Real-Time PCR

Changes in mRNA expression levels occur during physiological and pathological processes in the cardiovascular system. An increase inDNAtranscription results in increasedmRNAlevels and will subsequently result in increased protein levels that regulate processes inside and outside the cell. To determine alterations in mRNA levels, traditional methods such as Northern blot and ribonuclease protection assay can be used; however, large amounts of RNA are necessary and the methods are very labor intensive. In this chapter, we focus on the newest advancements in reverse transcription polymerase chain reaction (rt-pcr) technology, the real-time PCR or quantitative PCR, using small amounts of RNA to determine expression levels.We discuss the technique in general and describe two different approaches.