RNase Undetectable In Water After Ultrafiltration

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Examples
Quantitative RNase recovery at high flow
In one experiment, a polysulfone
Millipore Pyrogard UF membrane with a
nominal molecular weight cutoff of
13,000 Da was challenged with 200 liter
solutions containing 0.1 ng/ml of RNaseA
(Roche Diagnostics, 109 142) at a
throughput of 500 ml/min. This concentration
of RNase is between 10-100 fold
various water samples as measured by the
RNaseAlert assay. The table suggests that
RNase is ubiquitous in both treated and
untreated water samples. Even highly
purified water (reverse osmosis, Elix
water systems, unattended Milli-Q
Gradient systems) contains significant
RNase activity that could affect PCR
results. As expected, RNase-free, DEPCtreated
waters, and all UF-treated samples
Figure 2: Quantitative RT-PCR: Correlation between theoretical and experimental data.
higher than is normally encountered in most
PCR experiments. Total weight of collected
RNase A was 22 mg, which was essentially
quantitative (200 liters � 0.1 ng/ml).
RNase in sample waters
RNase activity was measured at various
stages of water purification with the
RNaseAlert test kit (Ambion), which
claims a lower detection limit of 0.003
ng/ml. RNaseAlert uses a cleavable,
RNase substrate standard labeled with a
green fluorescent probe. After generating
a calibration curve using an RNase standard
(Ambion), we tested samples on an
SFM 25 Konitron fluorometer, excited at
520 nm and read at 490 nm.
Table 1 lists RNase concentrations in
(Millipore Pyrogard D UF cartridge;
13,000 Da NMWL cutoff) showed the
lowest RNase levels,