Figure 5

Determination of relative abundances of EGFP and mouse Ass mRNAs during liver development by S1 nuclease mapping. (A) Strategy of S1 nuclease mapping. RNA structures, DNA probes and the predicted protected probe size after S1 nuclease digestion are depicted. Stars indicate ³²P 5'-end labels; wavy line at one end of the labeled probe indicates vector sequences that distinguish a re-annealed probe from a probe that was protected by hybridization to RNA. (B) Phosphorimage analysis of the protected fragments from S1 nuclease mapping. RNAs isolated from various stages of liver development (indicated on top) were hybridized to the 5' end-labeled probes and the DNA-RNA hybrids that resisted S1 nuclease digestion were electrophoresed through a 4% polyacrylamide gel containing 7 M urea. Gel was dried and analyzed by a phosphorimager. The protected fragments corresponding to mRNAs of mouse Ass, EGFP transgene and mouse Gapdh are marked on the right. Mouse lines from which RNAs were obtained are indicated on the left. (C) EGFP and Ass mRNA profiles during liver development. The intensities of hybridization signals of EGFP, Ass and Gapdh mRNAs were quantified by a phosphorimager where the levels of EGFP and Ass mRNAs were normalized to the level of the Gapdh mRNA. Relative abundances of EGFP mRNA (upper panel) and Ass mRNA (lower panel) during liver development are plotted by taking the stage of the highest level as 100%. Standard deviation (solid line) is shown if analysis was performed at least 3 times. If performed twice, average with interval of two values is shown (dotted line).