( he sample by theappearance of an app...)RNase Protection Assay (RPA),,,Using DIG-Labeled RNA Probes

he sample by the appearance of an appropriately sized probe fragment.

Factors Influencing the Assay

Transcript quality

• Long transcripts tend to give incomplete transcribed probes. We suggest the use of a relatively small probe for this assay (100300 nucleotides).
• RNase contamination
• Presence of transcription-termination signals in the template.
• Wraparound transcripts resulting from the use of REs that produce either a 3 overhang or a blunt end. It is strongly recommended to use a RE that produces a 5 overhang.
  
  

Quality of target RNA

• RNase contamination: When probe fragmentation is observed in the protection assay, check the integrity of your RNA sample by agarose electrophoresis.
• DNA contamination
  
  

Amount of sample and labeled probe

The sample size is usually in the milligram-range and the amount of labeled probe is around 500 pg1,000 pg.





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