Epidermal cell-specific quantitation of dopa decarboxylase mRNA in Drosophila by competitive RT-PCR: an effect of Broad-Complex mutants.
The quantitation of RNA in tissue homogenates by amplifying the product of reverse transcription (RT-PCR) is sufficiently sensitive to detect molecules in the range of 10(-1)-10(-2) amole. We describe here the steps we believe necessary to validate a protocol that used a DNA competitor and visualization of the amplification products by ethidium bromide staining. The procedure was designed to quantitate one of the tissue specific transcripts of the Dopa decarboxylase gene ( Ddc) in Drosophila. We demonstrate that the amount of epidermal Ddc transcript is much lower at pupariation in several mutants of the Broad-Complex, one of the primary response loci of the moulting hormone, ecdysone. The mutant effects were allele specific and the molecular basis of one of these alleles is known. This implicates a particular family of the zinc finger proteins encoded by the locus in the hormone dependent induction of Ddc expression.[1]References
- Epidermal cell-specific quantitation of dopa decarboxylase mRNA in Drosophila by competitive RT-PCR: an effect of Broad-Complex mutants. O'Keefe, S., Schouls, M., Hodgetts, R. Dev. Genet. (1995) [Pubmed]
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