Ambion's Tips From the Bench: Getting Rid of residual full length probe in ribonuclease protection assays

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Getting Rid of Residual Full Length Probe in Ribonuclease Protection Assays

When performing RPAs, we occasionally see remaining full length probe in both the experimental sample lanes and in the "+ RNase control" lane (probe + yeast RNA + RNase). While this can be a sign of incomplete RNase digestion, it can also result from residual template DNA that protects the full length size of the RNA probe. This explanation should be suspected when both full length probe and protected fragment bands are visible in the experimental samples and there is no smearing in between (smearing in between these two bands or below the full length probe band is usually indicative of incomplete RNase digestion).

Although a DNase step to remove DNA template is usually routine at the end of an in vitro transcription reaction, at Ambion we have empirically found that the newly synthesized RNA probe molecules can sometimes protect the complementary DNA template strand from digestion. We have shown this by labeling the DNA template prior to transcription, synthesizing unlabeled RNA, and then treating the reaction with DNase. Normally one would expect all of the labeled DNA template to have been digested. However when such a reaction is denatured, run on a polyacrylamide/urea gel, and exposed to film, a band of exactly the predicted probe size is seen. If such protection occurs, the researcher who gel purifies an RNA probe may still carry over some "protected DNA template" - those who don't gel purify certainly will.

To prevent carry over of template DNA:

If planning to gel purify in vitro transcribed RNA probes, skip the DNase step all together. The longer template DNA will not comigrate and therefore, will be left in the gel. Be sure to denature well before loading the gel. Otherwise, a small amount of probe may stick to the template and run double-stranded at a higher molecular weight.
Another option is to denature the template and newly synthesized RNA directly after the transcription reaction, before adding the DNase. We recommend doing this as follows:

Heat the transcription reaction to 95°C for 5 min.
Place immediately on ice.
Add DNase, bring to 37°C, and incubate 15-30 min.

Note: The denaturation step will also denature placental ribonuclease inhibitor (RIP) present in all of Ambion's transcription kits. If there is any RNase bound to the RIP, it will be released and become active.