The Use of LiCl Precipitation
for RNA Purification
LiCl has been frequently used to precipitate
RNA, although precipitation with alcohol and a monovalent cation
such as sodium or ammonium ion is much more widely used. LiCl
precipitation offers major advantages over other RNA precipitation
methods in that it does not efficiently precipitate DNA, protein
or carbohydrate (Barlow et al., 1963). It is the method of choice
for removing inhibitors of translation or cDNA synthesis from
RNA preparations (Cathala et al., 1983). It also provides a simple
rapid method for recovering RNA from in vitro transcription reactions.
Ambion provides LiCl as an RNA recovery agent
in its MEGAscript® and mMESSAGE mMACHINE® large scale in vitro
transcription kits. However, while providing telephone technical
service, we have noticed that many users are reluctant to use LiCl,
presumably because there is not good data in the literature describing
its properties. We have conducted a systematic study of the use
of LiCl and find that it is a very effective method for precipitating
RNA, especially from in vitro transcription reactions.
The three key variables we studied were: (a)
the temperature at which the precipitate is allowed to form, (b)
the concentration of the RNA and the lithium chloride used and,
(c) the time and speed of centrifugation used to collect the precipitated
RNA. All of these variables have been explored and are discussed
below. We find that LiCl precipitated RNA samples prepared in this
way require no further purification for use in hybridization and
in vitro translation reactions. It has been reported that lithium
chloride is unsuitable for cell free translations due to the inhibition
of chloride ions (Maniatis, et al., 1989); However, we have not
been able to document any deleterious effect in either translation
or microinjection experiments. Another advantage is that lithium
precipitation efficiently removes unincorporated NTPs, which allows
for more accurate quantitation by UV spectrophotometry.
Experimental Procedures
Unlabeled RNA transcripts with the lengths
of 100, 300, and 500 bases were synthesized in large amounts
using the MEGAscript in vitro Transcription Kit. Lithium chloride
was used to precipitate the RNA followed by resuspension in water.
The concentration of each RNA was determined by spectrophotometry.
An additional set of labeled transcripts were synthesized in
the presence of 50 µCi of alpha-[32P] UTP (800
Ci/mmol) to produce the three RNA transcripts with a specific
activity of 3.3 x 106 cpm/µg.
Comparison of Lithium Chloride and Ammonium
Acetate/Ethanol
In preliminary experiments, we compared
the precipitation efficiency of 2.5 M lithium chloride with 0.5
M ammonium acetate and 2.5 volumes of ethanol with RNA transcripts
of 100 and 300 bases in length. The average recovery with the
lithium chloride was 74% compared to 85% with the ethanol. Gel
analysis of the precipitated products suggested that the lithium
chloride may not precipitate the smallest RNA fragments as efficiently
as the ethanol. This can be an advantage when preparing labeled
probe for ribonuclease protection assays in that the lithium
chloride precipitated product will give a cleaner band on gel
analysis, especially with non-gel purified probe.
Precipitation Parameters of Lithium Chloride
RNA Concentration
Decreasing amounts of each size of RNA
were precipitated using a constant concentration of 2.5 M LiCl
to determine if there is a threshold of precipitation for a given
size and concentration of RNA. The three stocks of non-radioactive
RNA mixed with tracer labeled RNA (5 x 104 cpm) were
aliquotted in tubes. Water and then lithium chloride were added
to a final volume of 50 µl, with a constant concentration
of 2.5 M lithium chloride. Each size transcript was tested separately
to observe possible size effects on precipitation efficiency.
All samples were chilled 30 minutes at -20°C then centrifuged
for 15 minutes at 16,000 x g at 4°C. The supernatant was
removed by aspiration and dried for 10 minutes. The pellets were
resuspended in 10 µl of gel loading buffer (80% formamide,
0.1% bromophenol blue, 0.1% xylene cyanol, and 1mM EDTA) and
heated for 5 minutes at 95°C. A portion of each sample was
run on a 4% PAGE-urea gel. The gel was dried and exposed directly
to film for 30 minutes. Figure 1 shows the
effect of RNA concentration on lithium chloride precipitation
of the 100 base transcript. It appears that RNA as small as 100
nucleotides and as dilute as 5 µg/ml can be efficiently
precipitated by lithium chloride. This was a surprising result
since it is generally thought that RNA must be at relatively
high concentrations in order to be efficiently precipitated with
lithium chloride.
|
Figure 1. The
Effect of RNA Concentration on Lithium Chloride Precipitation. Lane
1, RNA size standards. Lane 2, 5µg/ml RNA, Lane
3, 50 µg/ml RNA, and Lane 4, 500 µg/ml of RNA. |
Lithium Chloride Concentration
The effect of lithium chloride concentration
on precipitation efficiency was tested on three different sized
transcripts. Each size transcript was kept at a constant concentration
of 1 µg/ml while the lithium chloride was tested at 2.5,
1.0, and 0.5 molar concentrations. Labeled RNA (5 x 104 cpm)
was also added as a tracer. The samples were centrifuged 10 minutes
at 4°C, aspirated and dried. The pellets were resuspended
in 10 µl of gel loading buffer, heated for 10 minutes at
95°C, and a portion of each was run on a 4% PAGE-urea gel.
The gel was dried and exposed for 30 minutes without intensifying
screens. Figure 2 shows the effect of lithium
chloride on precipitation of the 300 base transcript. It appears
that lithium chloride is effectively precipitating RNA at a 0.5
molar concentration and recovery was similar at all concentrations
of lithium chloride. Lane 5 is a zero lithium chloride control
to analyze the effect of centrifugation.
|
Figure 2. Effect
of Lithium Chloride Concentration on Precipitating RNA. Lane
1, RNA size standards. Lane 2, 2.5 M LiCl. Lane 3, 1.0
M LiCl. Lane 4, 0.5 M LiCl, and lane 5, no LiCl. |
Chilling Time
The RNA was kept at a constant concentration
of 1 µg/ml, with 1.0 M lithium chloride. The length of
time for precipitation was tested at 0, 0.5, and 1.0 hour. The
0.5 and 1.0 hour time points were incubated at -20°C and
25°C to test precipitation time and temperature independently.
Samples were prepared as before, and visualized on a 4% PAGE-urea
gel. In Figure 3, it appears that allowing
precipitation to occur for a 30 minute period is more efficient
than immediate centrifugation; compare Lane 2 to Lane 3. Although
it appears there is no difference in precipitating 30 or 60 minutes
at -20°C and 25°C, as seen in Lanes 3-6, it is advisable
to precipitate at -20°C for 30 minutes to lower the activity
of any possible RNases that might be present.
|
Figure 3. Effect
of Precipitation Temperature Using Lithium Chloride. Lane
1, RNA size standards. Lane 2, RNA centrifutged immediately
without chilling. Lane 3, RNA chilled at -20°C for
30 minutes before centrifugation. Lane 4, RNA incubated
at 25°C for 30 minutes to test precipitation time independently
of chilling. Lane 5, RNA chilled at -20°C for 1 hour.
Lane 6, RNA incubated at 25°C for 1 hour. |
Centrifugation Time
Using a constant concentration of 1 µg/ml
RNA, in a volume of 50 µl with 1.0 M lithium chloride,
samples were centrifuged for 0.5, 1, 2, 5, 10, and 20 minutes
at 4°C at 16,000 x g. The different sized transcripts, with
radioactive RNA, were tested independently. Figure
4 shows that centrifugation time is a major factor in recovery
of RNA. As little as 50 ng of RNA can be quantitatively recovered
by centrifugation at 16,000 x g for 20 minutes at 4°C. Lanes
2-7 show decreasing recovery as spin time is lowered.
|
Figure 4. Effects
of Centrifugation Time in Precipitating RNA. Lane
1, RNA size standards. Lane 2, RNA centrifuged for 20 minutes,
Lane 3, 10 minutes, Lane 4, 5 minutes, Lane 5, 2 minutes,
Lane 6, 1 minutes, and Lane 7, 30 seconds. |
Discussion
The use of lithium chloride in RNA precipitation
is a fast, convenient method of isolating transcripts from in
vitro transcription reactions with very low carry over of unincorporated
nucleotides. A major advantage of lithium chloride is that it
does not efficiently precipitate either protein or DNA. For some
applications, gel purification may be necessary, as in a ribonuclease
protection assay. For in vitro or in vivo translation, the lithium
chloride method may be preferable to ethanol precipitation since
full-length transcripts are often preferentially recovered. Moreover,
RNAs precipitated by this method give more accurate values when
quantitated by UV spectroscopy since lithium chloride is so effective
at removing free nucleotides. This strategy is similar to the
use of isopropanol rather than ethanol to precipitate nucleic
acids. Isopropanol is less efficient than ethanol at precipitating
nucleotides and thus, gives more accurate values when RNA concentration
is quantitated by UV spectrophotometry.
Contrary to previously published reports, we
find that lithium chloride does not appear to preferentially precipitate
higher molecular weight RNA rather than smaller RNA. Lithium chloride
precipitations using mixtures of equal amounts of RNA of lengths
100, 200, 300, 400, and 500 bases (RNA Century Size Standards)
showed that all sizes were precipitated equally well (data not
shown). Since it was thought that the larger sizes might aid in
the precipitation of smaller size transcripts, the experiments
in this paper were performed using each size of transcript separately.
No differences in precipitating a single size of RNA (e.g. 100
bases) as compared to a mix of all sizes of the RNA markers was
seen. It should be noted, however, that some small RNAs such as
tRNAs are not efficiently precipitated by lithium chloride. This
is likely due to the high degree of secondary structure in tRNA.
While we recommend the routine use of lithium chloride for precipitating
RNA from solutions containing at least 400 µg/ml RNA, we
are cautious about recommending its use with lower concentrations
of RNA until we have tested its use with a wider range of RNAs.
References
- Barlow, J.J., Mathias, A.P., Williamson,
R., and Gammack, D.B., (1963). A Simple Method for the Quantitative
Isolation of Undegraded High Molecular Weight Ribonucleic Acid. Biochem.
Biophys. Res. Commun. 13:61-66.
- Cathala, G., Savouret, J., Mendez,
B., West, B.L., Karin, M., Martial, J.A., and Baxter, J.D.,
(1983). A Method for Isolation of Intact, Translationally Active
Ribonucleic Acid. DNA 2:329-335.
- Maniatis, Sambrook, Fritsch, (1989). Molecular
Cloning: A Laboratory Manual 2nd ed., Vol. 3, Appendix
E.12.
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