Figure 1

Detection of Pax6 and Pax6(5a). (A) Riboprobe design. The ssRNA antisense riboprobe used to detect both Pax6 and Pax6(5a) spans the Pax6 mRNA from exon 3 to exon 5a. Two protected fragments are produced, Pax6 (218nt) and Pax6(5a) (247nt). The PD- and HD-coding regions are represented above the Pax6 mRNA sequence, which is shown to scale. Grey and black bars are exons. Broken line shows part of the riboprobe derived from the vector. (B) Example of an RNase protection assay showing GAPDH and Pax6 signal detected in total RNA from E12.5 eye (E), diencephalon (D) and telencephalon (T). 1 μg total RNA was used in each sample. Central lane is a DNA ladder, which allows approximate sizing of bands (fragment sizes are indicated above bands). (C) Quantification of RNase protection assays. Pax6 (lower band) and Pax6(5a) (upper band) in the telencephalon, diencephalon and eye at E12.5 and E18.5. Gel images and densitometric traces of bands are shown. Level of background estimated using the rolling disk method (Quantity One software, Biorad) is indicated on each trace.