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.
O'Keefe
S
S
Department of Genetics, University of Alberta, Edmonton, Canada.
Schouls
M
M
Hodgetts
R
R
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D004296:Dopa Decarboxylase / Q000235:genetics*; D004331:Drosophila melanogaster / Q000235:genetics*; D000078404:Epidermal Cells; D004817:Epidermis / Q000201:enzymology*; D017344:Genes, Insect; D008297:Male; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D012333:RNA, Messenger; D004296:Dopa Decarboxylase