Fig3-5.6.3: Labeling RNA probes

Riboprobes are mostly created by run-off transcription in which RNA insert is cloned in specialized plasmid vectors. The 5' terminus of the transcript is fixed by bacteriophage promoter, but 3' terminus is defined by downstream site of cleavage by restriction enzyme. Restriction enzymes that generate blunt or 5'-protuding termini produce best linear templates. Enzymes generating protruding 3' termini, should be avoided because transcription of such templates results in synthesis of RNA molecules that are aberrantly initiated at the termini of templates.

Commonly used systems for generation of riboprobes are Bacteriophage T3 and T7 promoter/RNA polymerase systems. Labeled riboprobes, both sense and antisense c an be generated from any gene cloned in such vectors (in either of the two orientations). These are widely used in tissue in situ hybridization.

The DNA fragment to be transcribed can also be amplified in PCR with primers having 5' ends encode synthetic promoters for bacteriophage RNA polymerases. Following purification, the PCR products can be used as double stranded templates for in-vitro transcription.