Fig. 3. Expression of GFR1 and Ret mRNAs, but not of GFR2 mRNA, is down-regulated in ciliary ganglion neurons during development. (A) Calibration PCR reaction profile with E5 ciliary ganglion neuron cDNA. To check the linearity of the detection system, various dilutions (1, 1:10, 1:100, 1:1000) of E5 ciliary ganglion neuron cDNA as well as water (control) were amplified with a primer pair and a TaqMan probe for L27 housekeeping gene. The correlation coefficient was calculated from the standard curve that displays threshold cycles (C_t) as a function of log₁₀ cDNA concentrations (inset). (B,C) Real-time PCR reactions for GFR1, GFR2 and Ret mRNA expression in E5 and E15 ciliary ganglion neurons. Real-time PCR for each of the three receptors was run using E5 (B) or E15 (C) ciliary ganglion neuron cDNA samples. For an internal control, the L27 mRNA level was measured with each cDNA sample. Each solid line in the graphs shows the average of quadruplicate PCR reaction profiles. Note that the Y axis in the graphs is in a log scale. A dotted horizontal line in each graph indicates a threshold for that experiment. (D-F) Comparison of the expression of GFR1, GFR2 and Ret mRNAs between E5 and E15 ciliary ganglion neurons. Based on Ct values obtained from 2 separate experiments, the mRNA level for each probe relative to L27 mRNA level (internal control) was calculated. The GFR1 and Ret mRNA levels in E5 ciliary ganglion neurons are approximately 19 times and 1.5 times higher, respectively, than those in E15 ciliary ganglion neurons. **, Significantly different from the E5 group (P<0.01); *, P<0.05.