PROTEIN-TYROSINE-PHOSPHATASE GENE-EXPRESSION ANALYSIS IN SWISS 3T3 FIBROBLASTS

The aim of this study was to identify protein tyrosine phosphatases (P TPs) expressed in Swiss 3T3 fibroblasts and to examine their expressio n levels as well as to characterize quantitative aspects of RT-PCR bas ed on degenerate deoxyoligonucleotides. By using an RT-PCR assay based on degenerate deoxyoligonucleotide primers, expression of mRNAs for t wo cytoplasmic- and six transmembrane-type PTPs in Swiss 3T3 cells was detected. The sequences of two of them are new. Among nine analyzed P TPs expressed to widely varied extends, only three have mRNA levels hi gh enough to be seen on Northern blots with 10 mu g of total RNA per l ane. The frequencies with which the examined PTPs are represented amon g the PCR amplification products, correlate stronger with the primer f idelity, defined as the number of mismatches between the primer- and t he cDNA target-sequences, rather than with the PTP expression levels. In conclusion, an RT-PCR assay based on degenerate primers can be succ essfully used to sample the expressed PTPs and to identify new members of this gene family. However, reliable quantification of their mRNA l evels can only be achieved using the classical approaches, like Northe rn, RNase protection assay or nondegenerate quantitative RT-PCR.