Rapid assessment of protein-tyrosine phosphatase expression levels by RT-PCR with degenerate primers
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Using degenerate oligodeoxynucleotide primers we previously obtained cDNA fragments from ten different murine protein-tyrosine phosphatases (PTPases). Employing this same primer set, a method was developed to assess the expression levels of these PTPase family members in a fast and simple way. RT-PCR products of several cell types and tissue samples were used as probes on dot-blots containing the ten different PTPase fragments in equimolar amounts. Hybridization intensities at the various dots reflect the relative expression levels of the corresponding PTPases in the starting material. In this way expression of PTPases during mouse brain development could be monitored. Expression of PTPδ was found to be absent in embryonic stem cells but high in fetal and adult brain. PTPɛ expression is shown to gradually increase in brain during maturation. Our method is generally applicable to gene families of which the transcripts can be detected with a single degenerate primer pair and is especially useful in situations where only limited amounts of RNA can be obtained.
Key wordsexpression polymerase chain reaction protein-tyrosine phosphatases reverse transcription signal transduction
polymerase chain reaction
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