Figure 1

Scheme of Reverse RNase Protection Assay (RePro) procedure. (1) Isolated organelles (after quantification) are immediately used for (2) in vitro transcription in the presence of labeled nucleotides (indicated by A, C, G, U). During in vitro transcription (10 min usually), newly synthesized RNAs that arise from either transcript initiation or elongation of RNAs initiated before chloroplast isolation are labeled by incorporation of radioactive nucleotides (asterisks). (3) The reaction is terminated by the addition of stop buffer and the RNA is subsequently isolated. (4) The required amount of in vitro generated asRNA (about 0.1 μg) is added to the chloroplast RNA and both RNAs are co-precipitated (5). The RNA pellet obtained is dissolved in hybridization buffer and hybridized at 64°C (6). (7) To remove none-hybridized, single-stranded RNAs RNase treatment is performed and the samples are subsequently analyzed on 4% polyacrylamide/8 M urea gels (8) according to the standard RNase Protection Assay protocol (RPA).