Consequences of Too Much Probe - Ambion
One of the most common mistakes in designing
a ribonuclease protection experiment is the addition of too much
RPA requires just enough to be in molar excess of the target mRNA
since the hybridization is done in such a small volume (e.g.
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
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
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
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
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.