Quantitation of digoxigenin-labeled DNA hybridized to DNA and RNA slot blots.
Quantitation of message from low-abundance mRNAs and limited availability of tissues requires sensitive methods for probe detection, accurate methods for quantitation of signal, and the ability to strip and reprobe membranes. A random-primed, digoxigenin-labeled probe from cDNA of FMO1, an isoform of the flavin-containing monooxygenase gene family, from rabbit was used in the evaluation and optimization of the Genius system for quantitation of signal from DNA and RNA slot blots. Criteria for optimization were a low signal to noise ratio, a linear increase in density of signal vs nuclei acid concentration of bands on X-ray film, complete stripping of membranes, and reproduction of the initial banding pattern upon rehybridization. A low signal-to-noise ratio was obtained with an aqueous prehybridization/hybridization solution. DNA slot blots were successfully quantitated before and after alkaline stripping from positively charged membranes. RNA slot blots were subject to excessive and uneven loss of RNA from the membranes during stripping procedures. Reliable quantitation for more than one cycle of detection required highly charged nylon membranes, and careful tailoring of RNA fixation methods and alkaline stripping conditions.
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000032:analysis; D018076:DNA, Complementary; D004076:Digoxigenin; D015151:Immunoblotting / Q000379:methods*; D007202:Indicators and Reagents; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D005810:Multigene Family; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010105:Oxygenases / Q000096:biosynthesis / Q000235:genetics*; D012313:RNA / Q000032:analysis; D011817:Rabbits; D015183:Restriction Mapping; D012680:Sensitivity and Specificity
D018076:DNA, Complementary; D007202:Indicators and Reagents; D015345:Oligonucleotide Probes; D012313:RNA; D004247:DNA; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D004076:Digoxigenin