Fig. 2. (A) Blots of RNA from early (pregastrulation) embryos (EE), mixed-stage embryos (ME), adult soma (AS) and adult total (AT) hybridized to four transcript-specific probes. EE: >90% pregastrulation embryos (Schauer and Wood, 1990). ME: mixed-stage embryos isolated by hypochlorite treatment of him-8(e1489) hermaphrodites. AS: glp-4(bn2ts) young adult hermaphrodites that had been shifted to non-permissive temperature (25°C) at hatching; these animals lack almost all germline cells (Beanan and Strome, 1992). AT: fem-2(b245ts) young adult hermaphrodites that had been shifted to non-permissive temperature (25°C) for 36 hours after hatching (the temperature-sensitive period), then returned to permissive temperature; these animals produce no sperm and therefore contain oocytes but no fertilized embryos (Kimble et al., 1984). Photographs of 18S rRNA UV absorption bands on the blots before probing are shown as loading controls (lower panels). The fourth (AT) lane of each gel is clearly overloaded relative to the other three. In preliminary experiments using equal specific activities for the 1a and 1b probes, the 1b bands were fainter than the 1a bands. In the experiment shown, a 1b probe at about twice the specific activity of the 1a probe was used in order to better compare band intensities among the four RNA preparations. (B) Results of real-time quantitative PCR experiments to compare the relative ratios of each transcript with total unc-62 mRNAs in the same RNA populations as analyzed in A. Figures in the table represent the ratio obtained for each primer pair relative to the ratio obtained for the EE sample, which was arbitrarily assigned a value of 1.0. The ranges shown for each ratio were calculated from the results for each triplicate set according to User Bulletin #2: ABI Prism 7700 Sequence Detection System (2001). This experiment was carried out three times with similar results; ratios and ranges shown in the figure are from one experiment.