RNase Undetectable In Water After Ultrafiltration

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I
Ultrafiltration
removes RNase
from water to below
detectable
limits.
RNase Undetectable In Water
After Ultrafiltration
Joseph Plurad and Stéphane Mabic
Introduction
Highly purified water has become a standard tool in molecular
biology. Techniques such as reverse osmosis, electrodeionization,
ultrafiltration, and chemical treatments provide water of varying
quality and utility. Water quality specifications differ for applications
such as liquid chromatography, in situ hybridizations, single
nucleotide polymorphism analysis, and DNA amplification.
Experiments involving reverse-transcriptase PCR (RT-PCR), plasmid
preparation, construction of cDNA libraries, and Northern blotting
minimally require RNase-free water. However, RNA preparations
from cells and tissues are complicated by the presence of
ribonucleases (RNases).
Water treatment
Autoclaving and heat treatment do not satisfactorily inactivate
RNase, which can survive being heated to 180 C for 4
hours. By killing bacteria, autoclaving promotes release
of bacterial RNases, which may regain significant
activity at experimental temperatures.
Treatment with diethylpyrocarbonate (DEPC),
an irreversible RNase inhibitor, has evolved as
the standard method for preparing RNase-free
water for PCR. DEPC reacts with an active-site
histidine residue on RNase, forming a carbamate
and liberating a molecule of carbon dioxide and
ethanol. Since the chemical reaction is irreversible,
RNase inactivation by DEPC is presumed to be complete
and permanent. Although vendors and end-users
employ DEPC to produce PCR-quality nuclease-free water, the
DEPC method suffers from several drawbacks.
As a chemical treatment, DEPC merely inactivates RNase without
removing it. Inactivated enzyme remains in solution. After a onehour
incubation, excess DEPC is destroyed by being heated to 121 C,
hydrolyzing pyrocarbonate to carbon dioxide and generating ethanol
as the side-product. Most of the ethanol evaporates from solution.
What remains of side products and spent reagent contributes significantly
to the water’s total organic carbon (TOC) content.
By contributing carbonate/bicarbonate, DEPC decreases resistivity
and lowers the solution’s pH, which may contribute to RNA
instability. As we have observed by monitoring DEPC-treated
water, inactivated RNase may regain some activity over time.
TOC content and conductivity value of various purified waters
from Millipore illustrates the potential unsuitability of DEPC-treated
water for sensitive experiments. DEPC-treated water from a
major vendor contained a TOC of 122,800 ppb and exhibited conductivity
of 2.9 µS/cm. Milli-Q water treated with DEPC was
somewhat better, with a TOC of 15,700 ppb, but even higher conductivity
than commercial DEPC-treated water, 3.4 µS/cm.
Although two other commercial RNase-free waters contained much
lower levels of TOC (821 and 361 ppb), they showed high conductivities
of 2.1 and 2.8 µS/cm, respectively.
Ultrafiltration (UF), the non-chemical alternative to DEPC treatment,
removes RNase from water to below detectable limits and
thereby removes, rather than contributing to, TOC. Typical UF
methods employ polysulfone UF membranes embedded in acrylonitrile
butadiene styrene polymer (ABS) housings. Neither membrane
nor housing materials contribute to product water’s ion or organic
content. Milli-Q water treated by ultrafiltration contained less than
15 ppb of TOC and conductivity of 0.055 µS/cm corresponding to
18.2 megohm cm resistivity.
Figure 1: Study of rRNA stability in different water types using agarose gel micro-electrophoresis and fluorescence detection (Agilent
Bioanalyzer 2100): DEPC-treated water, Ambion RNase-free water, and water purified by UF (13,000 Da NMWL cut-off membrane).
Bioscience TECHNOLOGY 11 • 2004

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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,