Advanced mRNA differential display: isolation of a new differentially regulated myosin heavy chain-encoding gene in...

In an effort to make mRNA differential display more amenable as a molecular screen, we have optimized the technology for the isotopic and non-isotopic detection of differentially regulated mRNAs. The number of amplification rounds in the displays was significantly reduced, resulting in the semi-quantitative detection of expression patterns of both low- and high-abundance transcripts. Moreover, we extended the method beyond the display of mRNAs by introducing a direct sequencing approach for the fast molecular analysis of isolated cDNAs. Applying this improved technique to the regenerating amphibian limb system, we have identified cDNA PCR products with a temporal difference in expression. This differential regulation was confirmed by Northern analysis, and DNA sequencing uncovered a novel newt differentiation-specific transcript encoding a skeletal myosin heavy chain (MHC).