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).

Abstract

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