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Applied and Environmental Microbiology, October 2000, p. 4547-4554, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The Presence of Humic Substances and DNA in RNA
Extracts Affects Hybridization Results
Elizabeth Wheeler
Alm,1,2,*
Dandan
Zheng,1 and
Lutgarde
Raskin1
Department of Civil and Environmental Engineering,
University of Illinois at Urbana-Champaign, Urbana, Illinois
61801,1 and Department of Biology,
Central Michigan University, Mount Pleasant, Michigan
488592
Received 7 April 2000/Accepted 19 July 2000
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ABSTRACT |
RNA extracts obtained from environmental samples are frequently
contaminated with coextracted humic substances and DNA. It was
demonstrated that the response in rRNA-targeted oligonucleotide probe
hybridizations decreased as the concentrations of humic substances and
DNA in RNA extracts increased. The decrease in hybridization signal in
the presence of humic substances appeared to be due to saturation of
the hybridization membrane with humic substances, resulting in a lower
amount of target rRNA bound to the membrane. The decrease in
hybridization response in the presence of low amounts of DNA may be the
result of reduced rRNA target accessibility. The presence of high
amounts of DNA in RNA extracts resulted in membrane saturation.
Consistent with the observations for DNA contamination, the addition of
poly(A) to RNA extracts, a common practice used to prepare RNA
dilutions for membrane blotting, also reduced hybridization signals,
likely because of reduced target accessibility and membrane saturation effects.
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TEXT |
Hybridizations with oligonucleotide
probes targeting rRNA extracted from environmental samples are commonly
used to characterize microbial communities (e.g., references
6, 10, 11, 17, and 21). When
membrane hybridization assays are used for the quantification of
populations in complex microbial communities, in particular for the
quantification of low-abundance populations, experimental conditions
need to be optimized for detectability and precision (13,
14). Since RNA extracted from environmental samples is frequently
contaminated with coextracted humic substances and DNA (3, 10,
11), it is necessary to evaluate the effects of the presence of
these contaminants on quantitative membrane hybridizations. We present
here results of experiments designed to accomplish this objective.
Humic substances are naturally occurring, heterogeneous organic
substances that are yellow to black in color, of relatively high
molecular weight, and resistant to degradation (1). They contain anionic functional groups (e.g., partially deprotonated phenolic and carboxylic groups), as well as hydrophobic components (aromatic and aliphatic moieties) (23). The coextraction of humic substances is particularly problematic when isolating RNA from
soils, sediments, and water (10, 11). Humic substances have
been shown to interfere with enzymatic digestion of DNA, with PCR
amplification of DNA (18, 22, 26) and, to a lesser extent,
with dot blot hybridization of DNA (24). Recent developments in nucleic acid extraction methods for environmental samples have focused on purification strategies to remove humic substances (7,
10, 27, 28). The effects of humic substances on RNA-targeted hybridizations have not, to our knowledge, been thoroughly addressed.
DNA can be present in RNA extracts in excess of the RNA recovered
(3). Contaminating DNA is often digested from RNA extracts with DNase (10, 11). A concern with this approach is that some DNase preparations contain residual RNase activity and cause partial degradation of the RNA (24). Nucleases affect
different regions of rRNA molecules to various degrees, and partial
degradation of the rRNA molecules may have a serious influence on
quantitative hybridization results (14). For example,
hybridization results with universal probes are generally used to
normalize the responses obtained with specific probes. Samples with
partial rRNA degradation of target sites for universal probes (e.g.,
the 1390 region in the small-subunit rRNA, Escherichia coli
numbering) may result in elevated responses (11, 14, 17).
Coextracted humic substances and DNA can be retained on hybridization
membranes by two possible mechanisms: (i) by binding to the membrane
directly or (ii) by interacting with RNA bound to the membrane. In the
first scenario, hybridization signals may be decreased since humic
substances and DNA occupy binding sites on the membrane, thus resulting
in fewer binding sites for rRNA (this phenomenon is referred to as
membrane saturation in this study). On the other hand, hybridization
signals may be increased due to nonspecific binding of the
oligonucleotide probes to the membrane-bound contaminants. In the
second scenario, humic substances and DNA bound to membrane-immobilized
rRNA may also result in an increased hybridization response due to
nonspecific binding of the oligonucleotide probes. It is also possible,
however, that they interfere with the hybridization of probes to target
rRNA, thus reducing hybridization signals.
To evaluate the effects of the presence of coextracted humic substances
and DNA on quantitative membrane hybridizations targeting rRNA, we
first determined the hybridization response with uncontaminated RNA. In
a previous study (13), it was demonstrated that Magna Charge, a 0.45-µm-pore-size charge-modified nylon membrane from Micron Separation, Inc. (Westboro, Mass.), exhibited better
detectability and lower variability for oligonucleotide probe
hybridizations compared to three other membrane types tested.
Therefore, Magna Charge membranes were used for all experiments in this study.
Hybridization response with uncontaminated RNA.
In a first
experiment, the hybridization response for immobilized RNA in the
absence of DNA or humic substances was evaluated for Magna Charge
membranes by hybridization with a 32P-labeled
oligonucleotide probe. RNA was extracted from a pure culture of
E. coli (harvested during exponential growth) using a
low-pH, hot-phenol method (16, 21). It was determined that DNA was not coextracted with RNA using polyacrylamide gel
electrophoresis (PAGE) (3) (data not shown). The RNA was
denatured, diluted to different concentrations (using double-distilled
water [ddH2O] containing 0.02 µl of 2% bromophenol
blue per ml), and applied in triplicate to a Magna Charge membrane by
slot blotting (15). Subsequently, the membrane was baked for
2 h at 80°C, hybridized with a 5'-end 32P-labeled
universal probe (S-*-Univ-1390-a-A-18 [30]), and
washed as previously described (15). The hybridized membrane
was exposed to a phosphor storage screen (Molecular Dynamics,
Sunnyvale, Calif.), which was scanned using a PhosphorImager (Molecular
Dynamics). The image was quantified with the software package
ImageQuant (Molecular Dynamics). Figure
1a shows the hybridization signals (expressed as the sum above background [SAB], the output obtained using ImageQuant) for the various amounts of RNA applied. The relationship between hybridization response and the amount of RNA
applied is not linear, especially for the higher amounts of RNA
applied. Figure 1b shows the change in slope between each one of two
sequential data points in Fig. 1a. The slope starts to decrease between
the datum points corresponding to 160 and 320 ng of RNA applied per
slot. Two mechanisms may be responsible for this observation. First,
the RNA binding capacity of the membrane may have been reached,
suggesting that the decrease in slope was due to membrane saturation.
Second, the accessibility of the target may have decreased when large
amounts of RNA were applied. Regardless of the mechanism(s) responsible
for the decrease in slope, a decrease in hybridization response appears
to take place between applications of 26.7 and 53.5 ng of
RNA/mm2 (the area of one slot is 6 mm2). For
the lower amounts of RNA applied (< 10 ng of RNA per slot), the slope
increases sharply. This behavior at low levels of radiation ("detectable reciprocity failure") is an artifact of the phosphor imaging technology (20).
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FIG. 1.
(a) Hybridization response for increasing amounts of RNA
(nanograms of E. coli RNA applied per slot), expressed as
SAB. (b) Change in slope between two sequential datum points in panel
a.
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For the experiments presented below, samples were diluted with
ddH
2O containing 0.02 µl of 2% bromophenol blue and 1 µg of
poly(A) per ml. Thus, since a sample volume of 50 µl was
applied
to each slot, 50 ng of poly(A) was added in addition to RNA,
DNA,
or humic acid. However, since the total amount of nucleic acid
applied per slot was <160 ng, membrane saturation and/or target
accessibility due to the presence of poly(A) should not be a concern
for the experiments described
below.
Effects of humic substances.
To evaluate the effect of the
presence of humic substances in RNA extracts on membrane
hybridizations, RNA samples extracted from pure cultures of
Methanosarcina acetivorans or E. coli (harvested during exponential growth) were mixed with various amounts of humic
acids (catalog no. h1,675-2; Aldrich, Milwaukee, Wisc.). The RNA-humic
acids samples were denatured, diluted with ddH2O containing
0.02 µl of 2% bromophenol blue and 1 µg of poly(A) per ml, and
applied to Magna Charge membranes (10 ng of RNA per slot) as described
above. The membranes were baked, hybridized, and washed using the
oligonucleotide probes listed in Table 1 (except for probe S-D-Euca-0502-a-A-16). The hybridization signals were
quantified as described above, and the results were expressed as a
percentage of the hybridization response for RNA samples that did not
contain humic acids (unamended).
To estimate the amounts of soil humic substances that might be
coextracted with 10 ng of RNA, the following assumptions were
made: (i)
1 g of soil contains 10
10 cells (
8,
10,
25), (ii) a typical soil bacterium contains
5.7 × 10
15 g RNA (
19), (iii) 80% of the
organic carbon content in soils
consists of humic substances
(
12), and (iv) all humic substances
present in a sample are
recovered during the extraction procedure.
Amounts of humic acids
corresponding to organic carbon contents
of 1% (freshwater sediment
[K. Nealson, personal communication],
1.4 µg of humic acids per 10 ng of RNA), 4% (coastal marine sediment
[
9], 5.6 µg
of humic acids per 10 ng of RNA), and 15% (soil
[A. Ogram, personal
communication], 21 µg of humic acids per 10
ng of RNA) were used in
this study. Note that the same four assumptions
developed for soils
were used for calculating the amounts of humic
acids to be added to
stimulate RNA extracts from freshwater and
marine sediments. The
results of this experiment were expressed
as a percentage of the
hybridization response for unamended RNA
samples and are shown in Fig.
2.
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FIG. 2.
Hybridization response for RNA amended with humic acids
and for humic acids alone. The hybridization response is expressed as a
percentage of the hybridization response obtained with RNA only.
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The hybridization signals obtained for the 21-µg humic acid (without
RNA) application ranged from 1.9 to 5.4% (average, 3.0%)
of the
hybridization responses obtained with 10 ng of unamended
RNA,
indicating that the oligonucleotide probes bind nonspecifically
to
humic acids to a low extent. The presence of 1.4 µg of humic
acids
did not have a significant effect on the hybridization signals
as
determined by a two-way analysis of variance (ANOVA) (
P < 0.05).
For humic acids amendments of >1.4 µg, the hybridization
responses
decreased (a 23.8% average decrease in the presence of 5.6 µg
of humic acids and a 62.9% average decrease for 21 µg of humic
acids). These decreases were significant as tested by a fixed
two-way
ANOVA (
P < 0.05) (factor 1 = probe, factor 2 = humic acid
level).
As discussed above, the decrease in hybridization signal due to the
presence of humic substances may be caused by two mechanisms,
membrane
saturation or interactions between humic substances and
RNA that affect
probe hybridization. To determine whether the
decrease in hybridization
signal was due to membrane saturation,
increasing concentrations of
humic acids were added to radiolabeled
RNA. Ten nanograms of
radiolabeled RNA, amended with various amounts
of humic acids, was
denatured, diluted with ddH
2O containing 0.02
µl of 2%
bromophenol blue and 1 µg of poly(A) per ml, and applied
to membranes
as described above. The membranes were baked, incubated
with
hybridization solution (without probe), and washed, and the
radioactivity was quantified as presented above. The results of
this
experiment (Fig.
3) showed that
radioactive signals were
reduced by 18.0, 36.6, and 63.0% for humic
acids amendments of
1.4, 5.6, and 21 µg, respectively. Since the
levels of signal
reduction for the humic acid amendments of 5.6 and 21 µg were
similar to those observed in the previous experiment, the
signal
decreases appeared to be due to membrane saturation. It is
unclear
why the amendment of 1.4 µg of humic acids to 10 ng of RNA
did
not have a significant effect on the hybridization response (Fig.
2), whereas the addition of the same amount of humic acids to
10 ng of
radiolabeled RNA resulted in an 18% decrease in radioactive
signal
(Fig.
3). It is possible that the nonspecific binding of
probes to
humic acids partially compensated the effects of membrane
saturation.
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FIG. 3.
Radioactive signal for increasing amounts of humic acids
added to 10 ng of radiolabeled RNA and for humic acids alone. The
signal is expressed as the SAB.
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In summary, the presence of humic substances in RNA extracts lowers
hybridization signals due to membrane saturation, i.e.,
by preventing
the total amount of RNA applied from binding to
the membrane. This
effect is less significant for humic acid amounts
of
1.4 µg per 10 ng of RNA applied to a membrane surface of 6
mm
2 (i.e., one
slot). On the other hand, small amounts of oligonucleotide
probes can
bind nonspecifically to humic substances, resulting
in a slight
increase in hybridization
signals.
Effects of DNA.
RNA extracts from environmental samples often
contain high levels of coextracted DNA relative to the amounts of RNA
recovered. For example, we determined that significant amounts of DNA
were coextracted during a low-pH, hot-phenol RNA extraction of samples obtained from a coastal microbial mat (29), an anaerobic
sewage sludge digester (16), and a solid waste digester
(6). The different nucleic acid fractions were visualized
after separation using PAGE (3) before and after exposure to
DNase (FPLCpure DNase 1; Pharmacia, Piscataway, N.J. [catalog no.
27-0514-01]) (Fig. 4a). The presence of
DNA in RNA extracts has not been considered during development of
rRNA-targeted hybridization techniques because DNA is generally absent
from pure culture RNA extracts, obtained using cells harvested during
exponential growth. However, when hybridization protocols are used to
quantify populations in environmental samples, in which cells are often
in their stationary growth phase (high DNA/RNA ratio), the effect of
the presence of DNA needs to be evaluated. To illustrate the difference
between DNA levels in RNA extracts obtained from cells harvested in the
exponential and stationary growth phases, we extracted RNA using a
low-pH, hot-phenol method from Methanosaeta concilii cells
harvested in exponential and stationary growth phases. The different
nucleic acid fractions were visualized after separation with PAGE (Fig. 4b).
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FIG. 4.
PAGE gels of nucleic acid samples extracted from various
environmental samples. (a) Lanes 1 and 2, coastal marine microbial mat;
lanes 3 and 4, anaerobic sewage sludge digester; lanes 5 and 6, solid
waste digester. Samples in lanes 1, 3, and 5 were treated with FPLCpure
DNase 1; samples in lanes 2, 4, and 6 are undigested controls. (b) Lane
1, Methanosaeta concilii 11 days after transfer into fresh
medium (exponential growth phase); lane 2, M. concilii 25 days after transfer into fresh medium (stationary growth phase). LSU
and SSU represent bands for the large rRNA of the large ribosomal
subunit (23S and 23S-like rRNA) and for the small-subunit rRNA,
respectively.
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To evaluate the effect of the presence of DNA in RNA extracts on
membrane hybridizations, RNA was removed from
E. coli DNA
(catalog no. D-2001; Sigma, St. Louis, Mo.) by RNase digestion
(Ribonuclease 1 A; Pharmacia). Subsequently, the RNase was
removed
by one phenol-chloroform extraction, one chloroform extraction,
and ethanol precipitation. Ten nanograms of RNA (without DNA)
obtained
using a low-pH, hot-phenol extraction from
E. coli harvested
during exponential growth was amended with 1, 10, or 100 ng of
the
RNase-treated DNA; denatured; diluted with ddH
2O containing
0.02 µl of 2% bromophenol blue and 1 µg of poly(A) per ml; applied
to membranes; hybridized; and quantified as described above. In
addition, hybridization results were obtained for 10 ng of RNA
(unamended) and 10 and 100 ng of DNA. The results were expressed
as a
percentage of the hybridization response for unamended RNA
samples and
are presented in Fig.
5.
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FIG. 5.
Hybridization response for RNA amended with DNA and for
DNA alone. The hybridization response is expressed as a percentage of
the hybridization response obtained with RNA only.
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In contrast to the hybridization signals obtained with humic acids
alone, the hybridization signals obtained with 10 and 100
ng DNA were
low (values ranged from 0.0 to 2.1% [average, 0.8%]
of the signal
obtained with 10 ng of unamended RNA), indicating
that oligonucleotide
probes did not bind appreciably to DNA for
the hybridization and wash
conditions used in this
experiment.
The effects of DNA contamination on hybridization signals were similar
to those observed for humic substances. The presence
of 1 ng of DNA did
not have a statistically significant effect
on the hybridization
signals as determined by a two-way ANOVA
(
P < 0.05).
As the DNA amendments increased to 10 and 100 ng,
the average
hybridization response decreased by 2.2 and 49.7%,
respectively. These
decreases were significant (
P < 0.05) as tested
by a
fixed two-way ANOVA (factor 1 = probe, factor 2 = DNA
level).
The possibility that the presence of DNA would result in membrane
saturation was examined in a manner similar to the experiment
described
above for humic substances. Increasing amounts of DNA
were added to 10 ng of
32P-labeled
E. coli RNA, and the nucleic
acids were denatured, diluted
with ddH
2O containing 0.02 µl of 2% bromophenol blue and 1 µg
of poly(A) per ml, and applied
to membranes, which were processed
as described above. The results of
this experiment are shown in
Fig.
6 and
confirm that DNA (at least for the levels used here)
did not prevent
the binding of radiolabeled RNA to the membrane
(the maximum reduction
in signal was observed for an amendment
of 10 ng of DNA and was only
6.7%). Since the level of signal
reduction due to the addition of DNA
to 10 ng of radiolabeled
RNA was much lower than the effect of DNA
contamination on hybridization
signals, the mechanism by which DNA
inhibits the hybridization
response does not appear to be related to
membrane saturation.
It is possible that the presence of DNA reduces
RNA target accessibility
due to interactions between DNA and RNA, but
further studies are
needed to confirm this hypothesis.
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FIG. 6.
Radioactive signal for increasing amounts of DNA added
to 10 ng of radiolabeled RNA and for DNA alone. The signal is expressed
as the SAB.
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Our observations that the presence of low levels of DNA decreased
hybridization signals and that membrane saturation and/or
reduced
target accessibility resulted in lower hybridization responses
for RNA
applied between 27 and 53 ng per mm
2 prompted us to
evaluate the effect of the addition of poly(A)
to the dilution water
used to prepare RNA samples. Historically,
poly(A) has been included in
dilution water to provide an alternative,
irrelevant target for
residual RNase activity. When RNA samples
are diluted before blotting,
the dilution water generally contains
1 ng of poly(A) per µl
(
15). Thus, since a 50- to 100-µl sample
volume is
blotted, 50 to 100 ng of poly(A) is applied with each
sample in
addition to the target RNA. To evaluate the effect of
the presence of
poly(A), dilution waters with three different
concentrations of poly(A)
(0, 1, and 5 ng/µl) were prepared. Various
samples with different
amounts of
E. coli RNA were denatured,
diluted with dilution
water, applied in 100-µl volumes to membranes,
and hybridized with
probe S-*-Univ-1390-a-A-18. Hybridization
signals were obtained as
described above and are plotted in Fig.
7. The inset in Fig.
7 shows that the
application of 100 ng of
poly(A) per slot, together with low amounts of
E. coli RNA (
40
ng), resulted in lower hybridization
signals than those obtained
with
E. coli RNA without
poly(A). Since the total nucleic acid
amounts in these samples were
below the earlier determined amount
of RNA that exhibited reduced
hybridization responses (about 160
ng per slot or 27 ng per
mm
2), a mechanism other than membrane saturation must have
been responsible
for the decreased response. As hypothesized above, it
is possible
that the presence of DNA [poly(A)] reduced RNA target
accessibility.
For higher amounts of
E. coli RNA (>40 ng)
and for the poly(A)
amendment of 500 ng per slot, the reduction in
signal was more
pronounced and may be explained by membrane saturation
effects.
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FIG. 7.
Hybridization response for increasing amounts of RNA,
with or without poly(A) in the dilution water. The hybridization
response is expressed as the SAB for increasing amounts of RNA applied
to the membranes (nanograms of E. coli RNA applied per
slot).
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A logical solution to problems associated with high levels of
coextracted DNA would be removal of DNA from RNA extracts by
DNase
digestion. Because of earlier reported concerns with residual
RNase
activity in DNase preparations, we evaluated the effect
of a DNase
digestion on hybridization response. Aliquots of pure
culture RNA
(Table
1) were exposed to two commercial RNase-free
DNase preparations
(RNase-free DNase 1, catalog no. 2222 [Ambion,
Austin, Tex.];
FPLCpure DNase 1 [Pharmacia]). The DNase was removed
by one
phenol-chloroform extraction, one chloroform extraction,
and ethanol
precipitation. Then, 10 ng of DNase-exposed RNA was
denatured, diluted
with ddH
2O containing 0.02 µl of 2% bromophenol
blue and
1 µg of poly(A) per ml, applied to membranes, and hybridized
as
described above. Hybridization results obtained with domain-specific
probes were expressed as a percentage of the hybridization response
obtained with the universal probe. The relative hybridization
responses
obtained with the RNA samples exposed to the DNase preparations
were
increased by 3.5 to 14.2% compared to controls not exposed
to DNase
(Fig.
8). This result may be explained if
the presence
of small amounts of RNase caused partial degradation of
the RNA
(e.g., the target site of the universal probe). If so, this
explains
the increase in relative hybridization response observed in
Fig.
8, since the universal probe was used to normalize the responses
obtained with the domain-specific probes.
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FIG. 8.
Hybridization response for RNA exposed to DNase. The
hybridization response is expressed as a percentage of the response
obtained with probe S-*-Univ-1390-a-A-18.
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Discussion and concluding remarks.
Contaminating humic
substances and DNA alter the results of quantitative membrane
hybridizations by lowering the expected hybridization response. The
presence of humic substances or high levels of DNA in RNA extracts
likely results in saturation of hybridization membranes, which
decreases the amount of target rRNA that is able to bind to the
membranes. The presence of low levels of DNA does not contribute much
to saturation but still reduces hybridization responses. This may be
due to interactions between contaminating DNA and target rRNA,
rendering target sites less accessible to oligonucleotide probes.
Removing contaminating DNA by digestion with DNase should not be
performed in most cases since DNase digestion may cause site-specific
degradation, resulting in elevated specific responses. If large amounts
of DNA contaminate an RNA sample, so that membrane saturation becomes a
problem, then DNase treatment may be necessary. However, the
consequences of site-specific degradation need to be taken into
account. The addition of poly(A) to dilution water can also decrease
hybridization signals, apparently due to mechanisms similar to those
observed for native DNA, and is not recommended if the total amount of nucleic acids [target plus poly(A)] exceeds the binding capacity of
the membrane.
When performing quantitative membrane hybridizations of environmental
samples, normalizations of specific probe results with
universal probe
results are necessary. The data presented to date
that have not been
normalized should be viewed with caution. Since
the decreases of
hybridization signals due to membrane saturation
by humics or DNA are
fairly uniform for different target sites,
this normalization approach
should help to reduce biases caused
by contamination by humic
substances and DNA. In situations where
target RNA has been subjected
to site-specific degradation by
RNase, however, normalizations will
result in falsely elevated
specific responses. Instead, greater care
should be taken in obtaining
RNA extractions without contamination by
DNA, rather than relying
on subsequent DNase treatment. Methods for
removing contaminating
humic substances and DNA from environmental RNA
extracts, without
causing (partial) degradation of rRNA, are needed to
yield high-quality
quantitative membrane hybridization
results.
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ACKNOWLEDGMENTS |
We thank Katherine McMahon for help with preparing Fig. 4, Scott
McNaught for help with statistical analyses, and Dominic Frigon and
Daniel Oerther for critical review of the manuscript.
This work was supported by grants from the U.S. National Science
Foundation (BES 9410476) and from the U.S. Department of Agriculture
(95-37500-1911).
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biology, Central Michigan University, 157 Brooks Hall, Mount Pleasant, MI 48859. Phone: (517) 774-2503. Fax: (517) 774-3462. E-mail: Elizabeth.W.Alm{at}cmich.edu.
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Applied and Environmental Microbiology, October 2000, p. 4547-4554, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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