Preparation of Probe

DIG-labeled riboprobes are synthesized by the transcription of sequences of interest which have been cloned into a vector such that they are flanked by two different RNA polymerase binding sites. The vector is first linearized with a restriction enzyme such that transcription produces "run-off" transcripts, that is, transcripts derived from the insert sequence alone. The restriction enzyme is chosen such that transcription yields an RNA probe which is complementary (antisense) to the target mRNA. The probe transcribed from the opposite strand (sense probe) can be use as a negative control.

The length of the probe is important. Probes of up to 1 kb are optimal. Longer probes penetrate the tissue less efficiently but can be partially degraded by alkaline hydrolysis to a more suitable size (4).

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