Detection of differentially expressed gelatinase A in metastatic and non-metastatic subpopulations of tumor cells by target RNA arbitrarily primed polymerase chain reaction (TRAP-PCR)

We have developed a novel procedure called Targeted RNA AP-PCR (TRAP-PCR) t o quantitatively measure specific mRNA expression. The target mRNA is rever se transcribed using a specific primer and PCR is performed under low strin gency conditions to generate a rich fingerprint-type band pattern. In this situation multiple sequences are coamplified with the targeted sequence. Th e amplification is carried out in a competitive fashion and is, in conseque nce, quantitative. We have applied this technique to determine Gelatinase A (GeI A) mRNA expression in the MXT mouse mammary carcinoma system. TRAP-PC R analysis using primers for Gel A produced a reproducible fingerprint incl uding one major band whose identity was confirmed to be Gel A cDNA, Highly metastatic MXT subclones show an increased Gel A expression. Results were c onfirmed by Northern blot and protein activity (gelatin zymography), TRAP-P CR is a simple, sensitive and specific technique to comparatively quantify mRNA expression and requires less template than conventional methods.