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. 2010 Aug 24;5(8):e12315. doi: 10.1371/journal.pone.0012315

Figure 6. Ribonuclease protection assays.

Figure 6

A. Diagram showing RPA strategy, with Math5 cDNA, two different antisense cRNA probes, protected fragments expected for FL (full length, unspliced) and ECO (spliced) transcripts, and positive control RNAs generated by sense IVT reactions. B. Autoradiogram, showing undigested probes A and B (366 nt and 632 nt) and exclusively unspliced fragments protected by E14.5 eye RNA (567 nt and 301 nt). No fragment corresponding to the presumptive ECO transcript (212 nt) was protected by eye RNA using either cRNA probe, although a doublet of this size was protected by the ECO IVT positive control. Background fragments observed with probe B (arrowheads) are caused by intrinsic sensitivity of the cRNA-mRNA duplex to RNase cleavage at particular sites and were also present in the full length IVT positive control. The probe (no RNase) and IVT controls were diluted 20- and 10-fold respectively, compared to the E14.5 eye RNA hybridization lanes.