Protein tyrosine phosphatase gene expression analysis in Swiss 3T3 fibroblasts.
The aim of this study was to identify protein tyrosine phosphatases (PTPs) expressed in Swiss 3T3 fibroblasts and to examine their expression levels as well as to characterize quantitative aspects of RT-PCR based on degenerate deoxyoligonucleotides. By using an RT-PCR assay based on degenerate deoxyoligonucleotide primers, expression of mRNAs for two cytoplasmic- and six transmembrane-type PTPs in Swiss 3T3 cells was detected. The sequences of two of them are new. Among nine analyzed PTPs expressed to widely varied extends, only three have mRNA levels high enough to be seen on Northern blots with 10 microg of total RNA per lane. The frequencies with which the examined PTPs are represented among the PCR amplification products, correlate stronger with the primer fidelity, defined as the number of mismatches between the primer- and the cDNA target-sequences, rather than with the PTP expression levels. In conclusion, an RT-PCR assay based on degenerate primers can be successfully used to sample the expressed PTPs and to identify new members of this gene family. However, reliable quantification of their mRNA levels can only be achieved using the classical approaches, like Northern, RNase protection assay or nondegenerate quantitative RT-PCR.
Celler
J W
JW
Max Planck Society Research Unit Molecular Cell Biology, Medical Faculty, Friedrich Schiller University in Jena, Germany.
Luo
X
X
Böhmer
F D
FD
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D005347:Fibroblasts / Q000201:enzymology; D015870:Gene Expression; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017027:Protein Tyrosine Phosphatases / Q000235:genetics*
D017931:DNA Primers; D009841:Oligonucleotides; D017027:Protein Tyrosine Phosphatases