RNase Protection Assay (RPA),,,Using DIG-Labeled RNA Probes ( 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.

Source:

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