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Purification of Rna

Purification of Rna

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06/12/2011

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Chapter 4
Purification of RNA
Miles Wilkinson
Microbiology and Immunology Department and the Vollum Institute. OregonHealth Sciences Laboratory. Portland
OR
97201.
USA
Additional material provided
by
T.A.
Brown
1 Introduction
The most important consideration
in
the
preparation
of
RNA
is
to rapidly andefficiently inhibit
the
endogenous ribonucleases which are present
in
virtuallyall living cells. There are several classes
of
ribonucleases which have
been
wellcharacterized
in
eukaryotic
and
prokaryotic organisms, including
both
endonucleases
and
exonucleases. Methods designed for
the
preparation
of
RNA
depend
on
speed and/or efficiency
of
neutralization
of
ribonucleases for
their
success. Most methods stipulate
the
use
of
ribonuclease inhibitors such
as
RNasin,vanadyl ribonucleoside complexes, guanidinium hydrochloride, guanidiniumisothiocyanate, heparin,
and
dextran sulphate, to
name
a few. Proteinase K
is
sometimes used to degrade ribonucleases,
and
organic solvents such
as
phenol
and
chloroform are used
to
remove ribonucleases by extraction procedures.Another important concern
in the
preparation
of
RNA
is
to avoid accidentalintroduction
of
trace amounts
of
ribonucleases from hands. glassware andsolutions. Finally, it
is
desirable
that the
methods used for
the
preparation
of
RNA
are relatively rapid
and
simple. Literally hundreds
of
procedures for
RNA
preparation exist,
many
of
which are time-consuming and labour-intensive. Themethods described here are relatively simple to perform
and
provide intact
RNA
suitable for
cDNA
cloning.
RNA
(Northern) blotting.
or
in vitro
translation. Techniques for
the
preparation
of
total cellular
RNA
from animal and plant cells.lower eukaryotes and prokaryotic cells are provided. Several alternative methodsare given which allow for
the
purification
of
cytoplasmic
RNA.
nuclear
RNA,
and
poly(A)+
RNA
from eukaryotic cells. A rapid mini-prep method for
the
isolation
of
cytoplasmic
RNA
from small numbers
of
eukaryotic cells
is
also provided.Techniques to prepare polysomal
RNA
and immunoprecipitate specific polysomal
mRNAs
are described elsewhere
(1-3).
2 Ribonuclease-free conditions
It
is
worth
devoting time towards
the
task
of
making a 'ribonuclease-free environment' in
part
of
your laboratory. This includes setting aside
an
area
of
the
69
 
MILES WILKINSON
70
laboratory for reagents
and
utensils to be used specifically for
RNA
work.
It
is
desirable, for example, to use a particular colour tape to label ribonuclease-freesolutions. Autoclaving effectively destroys
many
ribonucleases which may bepresent
in
laboratory solutions,
but
autoclaving does
not
inactivate some classes
of
ribonucleases, which
is
why
RNase
A,
for example,
is
rendered DNase-freeby boiling (see
Appendix
2).
Most reagents used for
RNA
work can be maderibonuclease-free by treating
them
with
DEPC,
a strong,
but not
absolute inactivator
of
most ribonucleases
(4).
Because
DEPC
can modify
RNA
molecules (bycarboxymethylation) and efficiently inactivate most enzymes. it
is
critical
to
deplete all traces
of
DEPC
before using a treated solution.
DE PC
is
removed byhigh temperatures which converts
it
to ethanol
and
CO
2.
The following
is
a list
of
precautions for preparing ribonuclease-free laboratory materials.
(1)
Use
gloves for all
work
involving
RNA.
(2)
Bake glass bottles and all
other
glassware for
at
least
4h
at
250°C. Incubate
the
bottle tops
in
0.1%
(v/v)
DEPC
in
water for
at
least
4h
at
37°C. replace
the
tops
on the
bottles and autoclave for 15
min
to inactivate
the
DEPC.
Caution:
open
the
DE PC
container in a fume hood.
(3)
Most plasticware. including conical tubes
and
pipettes. are relatively ribonuclease-free and hence
can
be used
without
any special preparation.
(4)
Autoclave pipette tips in their original containers before use. Microfuge tubesshould be transferred
to
ribonuclease-free containers (without directly handling
the
tubes) and autoclaved. The tubes are removed for use
with
forceps
and
carefully handled so
that
the
top
of
the
tube does
not
touch
the
bench
or
gloved hands.
(5)
Ribonuclease-free water
is
prepared by adding
DEPC
to a final concentration
of
0.1%
to double-distilled water (or equivalent). Autoclave for 1 h, open
the
autoclave door briefly to release steam.
and
leave overnight
in the
autoclave(turned
off.
but
hot) to decompose residual
DEPC.
Alternatively. boil
the
waterfor
at
least
30
min
on a heating pad.
(6)
Ribonuclease-free solutions are prepared, whenever possible, by treating with
DEPC
as
described above.
(7)
Solutions containing the buffer Tris cannot effectively be DEPC-treated becauseTris inactivates
DEPC.
Sucrose cannot be DEPC-treated because autoclavingsucrose causes it to caramelize.Since
ammonium
acetate
is
relatively volatile,it also cannot be autoclaved. For reagents
in
this category.
it
is
recommended
that
they
are prepare using ribonuclease-free water. Remove
the
reagentsfrom
their
original containers by tapping gently into a plastic weighing boat.Avoid using spatulas. magnetic stirrers, or beakers unless they have
been
baked
at
250°C for
at
least
4h
to destroy ribonucleases. Autoclave
the
solutions for
at
least 30 min. Autoclaving alone will effectively inactivate
many
classes
of
ribonucleases which may contaminate reagents
in
trace amounts.
 
PURIFICATION
OF
RNA
3 Quantification of
RNA
RNA
is
accurately quantified by measuring its absorbance in a spectrophotometer. The
OD
of
RNA
is
measured
at
its
maximum
absorbance wavelength
of
260 nm. One
OD
unit
is
equivalent to 40
f-Lg/ml
of
RNA.
Typically, a small aliquot
of
a
RNA
sample
is
used for quantification
and
then
discarded. Use a dilution
of
the
RNA
sample which will give
an
OD
value
of
between
0.1
and
0.5
(2-10
f-Lg
in
a 0.5-ml cuvette). In cases where only small amounts
of
RNA
are being assessed,it may be more appropriate to save
the
RNA
after
OD
determination. In thiscase, set aside a cuvette which will only be used for nucleic acid (preferably only
RNA)
quantification. Before use, fill
the
cuvette
with
freshly prepared
0.1%DE PC
in
water and
incubate
at
least 10
min at
37°C, followed
by three
rinses
with
ribonuclease-free water.Contamination
of
RNA
preparations
with
substantial amounts
of
protein
is
demonstrated by measuring
the
OD
at
a wavelength
of
280 nm, as proteinstypically have a
maximum
absorbance
at
this wavelength. A 260-280
nm
absorbance ratio
of
1.8-2.0
is
appropriate for pure
RNA.
Contaminating proteinwill result
in
lower values
of
the
ratio (values below 1.6 indicate serious proteincontamination). Significant quantities
of
DNA
are rarely present
in
RNA
preparations made by
the
methods described
in
this chapter. However,
if
DNA
contamination
is
suspected (for example,
if
the
sample
is
extremely viscous
and
'stringy') this can be assessed by electrophoresing
the
sample
on
a denaturingagarose gel
and
staining with ethidium bromide, as described
in
Chapter
5,
Section
4.
DNA
cannot be distinguished from
RNA
by spectrophotometry since
both
exhibit a
maximum
absorbance
at
a wavelength
of
260 nm.
4 Precipitation
and
storage of
RNA
Like
DNA,
RNA
is
precipitated
in the
presence
of
alcohol
and
salt. A standardmethod to precipitate
RNA
is to add a
0.1
volume
of
3
M
sodium acetate
pH
5.2,2.0-2.5 volumes
of
ethanol, followed by vigorous mixing,
and
precipitation
at
-20°C. Studies
with
DNA
have indicated
that
precipitation
of
nucleic acids occursmost efficiently between
-20°C
and room temperature
(5),
and
notat
-70°C
asis
commonly believed. The length
of
ime
for precipitation
and
for centrifugationdepends
on the
amount
of
RNA
to be precipitated. For
RNA
samples
at
aconcentration
in
ethanol
of
greater
than
10
f-Lg/ml,
precipitate for
at
least 20
minat
-20°C,
and
centrifuge for 10
min at
room temperature. For
RNA
samples
at
aconcentration ofless
than
10
f-Lg/ml.
centrifuge for 30 min. Trace amounts
of
RNA
«
100 ng/ml) should be precipitated overnight to improve recovery.Generally,
RNA
is
centrifuged
in
1.5
ml
tubes in a microfuge
at
maximum
speed.
If
samples larger
than
1.5
ml
are
to
be centrifuged, several options areavailable:• Split
the
sample into multiple 1.5
ml
microfuge tubes• For
RNA
samples
at
a concentration
of
greater
than
10
f-Lg/ml,
pellet
in
15
or
50
ml
polypropylene conical tubes
at
2500 g
71

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