A METHOD TO MONITOR MESSENGER-RNA LEVELS IN HUMAN BREAST-TUMOR CELLS OBTAINED BY FINE-NEEDLE ASPIRATION

A method based on the reverse transcriptase-polymerase chain reaction (RT-PCR) was developed that allows the determination of relative mRNA expression levels in fine-needle aspirates from human tumors. The meth od was developed for the c-erbB-2 gene, using the porphobilinogen deam inase (PBGD) gene as an internal standard. It was validated for mRNA i solated from cell lines and for material obtained by fine-needle aspir ation from human breast cancer. Gene expression levels were determined by measuring the activity of radiolabeled RT-PCR-amplified gene-speci fic bands with a phosphor imager. At least four points are measured on the log-linear part of the amplification cycle versus signal intensit y curves, and subsequently the distance between the curves of the gene of interest and that of an internal standard gene is used to calculat e the relative expression levels. The method worked equally well with the BRCA1 gene, illustrating that it can be generalized to other genes . The method is suitable to measure or monitor semiquantitively gene e xpression levels in accessible human tumors in situ.