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)

Clin Exp Metastasis. 1998 Oct;16(7):603-11. doi: 10.1023/a:1006584910365.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Northern
  • Female
  • Gelatinases / genetics*
  • Mammary Neoplasms, Experimental / genetics*
  • Mammary Neoplasms, Experimental / secondary
  • Matrix Metalloproteinase 2
  • Metalloendopeptidases / genetics*
  • Mice
  • Neoplasm Invasiveness
  • Polymerase Chain Reaction / methods*
  • RNA, Messenger / metabolism
  • Sensitivity and Specificity
  • Tumor Cells, Cultured

Substances

  • RNA, Messenger
  • Gelatinases
  • Metalloendopeptidases
  • Matrix Metalloproteinase 2