Ribonuclease protection assays (RPAs) are used to measure discrete mRNA transcription levels from tissues or cell culture. This involves the use of an easily detectable antisense probe, which is complementary to part of a specific RNA species. After probe:RNA sample hybridization is allowed to occur, the sample is degraded with a single-strand specific nuclease to leave only the double-stranded "protected" fragments. The results are usually visualized on a polyacrylamide gel to resolve the size shift of the original probe and its digestion product.
Consequences of Too Much Probe
One of the most common mistakes in designing a ribonuclease protection experiment is the addition of too much probe. Those who have performed Northern blotting protocols understand the need for large amounts of probe. On the other hand, RPAs require just enough to be in molar excess of the target mRNA since the hybridization is done in such a small volume (e.g. 20 µl). It is true that adding more probe will enhance the hybridization kinetics and give a stronger signal. In other words, more probe gives a better protected fragment signal in the same amount of time than a sample with a lesser amount of probe. However, if the problem is examined from an enzyme kinetics view, as more and more probe is added, the substrate concentration will eventually rise above the Vmax of the nuclease. This will result in a failure of the enzyme's ability to destroy all unprotected probe within the standard digestion incubation period. In order to prevent this, the probe concentration must be kept below the Vmax of the nuclease. Empirically, this is approximately no more than 1-2 fmol of probe per sample tube. This is acceptable, as at these concentrations the probe is still saturating in relation to target (except when detecting ribosomal RNA) for amounts of up to 80 µg of total RNA sample. In addition, most of the RNA found in a total RNA extract is ribosomal and transfer RNA, which does not serve as substrate for the nuclease and does not compete against mRNA for hydrolysis. Adding more nuclease is not a fix, because it tends to either overdigest the protected fragment during digestion or before loading a gel, and thus only contributes to higher background.
How Much Probe to Add
- When synthesizing internally labeled RNA probe (using the MAXIscript™ Kit) with [alpha-32P] NTP (800 Ci/mmol, 10mCi/ml) as the limiting nucleotide, it is sufficient to use 1 x 104 cpm/10 µg total RNA. The upper limit is roughly 1 x 105 cpm/sample. If the specific activity of the probe is deliberately lowered by supplementing with "cold" NTP, proportionally less cpm are needed.
- When using nonisotopically modified RNA probe, 100 pg/10 µg total RNA is sufficient. The upper limit is 800 pg.