RNA probes are often made by transcription from a synthetic DNA template in vitro. These probes are similar to DNA probes with equal or greater binding affinity to homologous sequences. Because RNA and DNA form a stronger helix than DNA/DNA, the RNA probes may offer more sensitivity than DNA probes in the Southern blot.
RNA probes can be synthesized directly from a plas-mid template or from template DNA produced by PCR (see Chapter 7). Predesigned systems are commercially available for this purpose. These products include plas-mid vector DNA such as pGEM (Promega) or pBluescript (Stratagene), containing a binding site for RNA poly-merase (promoter) and a cloning site for the sequences of interest, and a DNA-dependent RNA polymerase from Salmonella bacteriophage SP6 or E. coli bacteriophage T3 or T7. DNA sequences complementary to the RNA transcript to be analyzed are cloned into the plasmid vector using restriction enzymes. The recombinant vector containing the gene of interest is then linearized, and the RNA probe is transcribed in vitro from the promoter. RNA probes are labeled by incorporating a radioactive or modified nucleotide during the in vitro transcription process.
Either coding or complementary RNA will hybridize to a double-stranded DNA target. Care must be taken, however, in designing RNA probes for Northern blots. The complementary sequence to the target must be used for the probe. A probe of identical sequence to the target RNA (coding sequence) will not hybridize. Because of labeling during synthesis, RNA probes can have a high specific activity (signal to micrograms of probe) that increases the sensitivity of the probe. To avoid high background, some protocols include digestion of nonhy-bridized RNA, using a specific RNase, such as RNase A, after hybridization is complete.
RNA probes are generally less stable than DNA probes and cannot be stored for long periods. Synthesis of an RNA probe by transcription from a stored template is relatively simple and should be performed within a few days of use. The DNA template can be removed from the probe by treatment with RNase-free DNase. Although RNA is already single-stranded, denaturation before use is recommended in order to eliminate secondary structure internal to the RNA molecule.
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