Monitoring MMP andTIMP mRNA Expression by RT-PCR

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) is a sensitive and rapid method to monitor the expression of specific mRNAs. Here we describe two approaches to quantification of steady-state levels of Matrix Metalloproteinase (MMP) and Tissue Inhibitor of Metalloproteinase (TIMP) mRNAs using RT-PCR. The first is a modified RT-PCR protocol called the “primer-dropping” method (1 ), which involves the simultaneous amplification of the specific MMP and TIMP target mRNAs (which are expressed in variable amounts in human cells) and that of an internal standard (glyceraldehyde phosphate dehydrogenase, GAPDH) which is expressed at constant levels. The internal standard provides a means to monitor the RT-PCR reaction efficiencies and to normalize the reaction products. The second approach uses coamplification of the specific target with a multi-MMP competitor cDNA within the same tube (2 ).