The RNase protection assay is a highly sensitive and reliable method for measuring RNA levels (Fig. 1), commonly used to measure circadian fluctuations of mRNA levels (see, for example, refs. 1-4). With exon-specific probes, it is also possible to study alternative splicings or the use of different promoters. Usually, the antisense riboprobes are about 100 to 200 nucleotides long. They can be placed at the junction between an intron and an exon, so that any trace of contaminating genomic DNA will result in a protected product of longer length than that obtained with the mRNA of interest. The riboprobes are radiolabeled during in vitro synthesis and hybridized to mRNA samples (see Fig. 1). The probe/mRNA hybrid is protected from RNase degradation, whereas the free probe is digested. A denaturing gel is then used to measure the amount of probe that was protected. It is the direct reflection of the amount of the RNA of interest that was present in the sample.

Fig. 1. Major steps of the RNase protection assay. Synthesis of the antisense riboprobe with an RNA polymerase (here T7 polymerase, T7pol), using as a template a linearized plasmid with a T7 promoter (T7p). Hybridization between the riboprobe and the RNA samples. RNase digestion: double-stranded RNAs are resistant to digestion. Denaturing polyacrylamide electrophoresis: Lane 1: radiolabeled molecular marker. Lane 2: untreated RNA probes. Lane 3: RNA probes without RNA samples, RNase treated (negative control; see Note 1). Lane 4: RNA probes hybridized to the template plas-mids and treated with RNases (positive control; see Note 1). Lanes 5-8: RNA probes hybridized to RNA samples collected at different zeitgeber times, hybridized to two probes (A and B; see Note 2) and RNase-treated. (A) Signals obtained with a probe directed against a gene under circadian regulation. (B) Signals obtained with a probe directed against a gene expressed at constant levels, which can be used to normalize the A signal of each sample (see Note 3). When hybridized to the RNA, the 5'-end of the probes are cleaved by the RNases, because this region contains plasmid-derived sequences that will not hybridize to the targeted RNA. Thus, they will run faster on the gel than the full-length probes.

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