New Nucleic Acid Techniques

554 Pages·1988·29.054 MB·English

by  John M. Walker

Chapter 1
Electrophoresis of RNA Denatured
with Glyoxal or Formaldehyde
Christopher F. Thurston, Caroline R. Perry,
and Jeffrey W.P ollard
1, Introduction
The first successful method for electrophoretic analysis of
the full size range of cellular RNA molecules was described by
Loening (I), and its introduction allowed for major advances,
most particularly in the molecular biology of eukaryotic organ-
isms. The method had, nevertheless, two significant disadvan-
tages in that the gels (composed of acrylamide at very low con-
centrations) were mechanically fragile, and the migration of
RNA molecules did not necessarily reflect their size because
RNA secondary structure was not disrupted.
More modern methods have consequently sought to in-
corporate conditions under which RNA is fully denatured and
that avoid the use of very fragile gels. Fragility of the gel was
overcome by use of agarose either in combination with, or in
place of, acrylamide. The problem of RNA denaturation is,
however, more complex. It is necessary both for analysis of the
2 Thurston,P erry,a nd Pollard
integrity of RNA samples, because secondary structure can
mask strand breaks to a significant extent, and because both
intramolecular and intermolecular interactions must be avoid-
ed if the size of RNA molecules is to be deduced from their rate
of migration during electrophoresis. In our view, glyoxal(2)
and formaldehyde (3) are the preferable denaturing reagents.
Theuseof formamide (4) or urea (5) involves more complicated
procedures without giving more thorough denaturation.
Methyl mercuric hydroxide is commonly cited as the most effi-
cient denaturant of RNA (6,7), but it is so toxic that its use can-
not be justified, except when no other method gives adequate
denaturation. Indeed, we have not seen anexample in the liter-
ature in which an RNA was denatured successfully by methyl
mercuric hydroxide and not by glyoxal or formaldehyde.
In this chapter we describe the separation of RNA on flat-
bed gels using only agarose (because the elimination of
acrylamide makes the procedure simpler and less hazardous
and has no significant disadvantages) and either glyoxal or
formaldehyde as denaturing agents. Both these methods pro-
vide RNA separation that is suitable for detection of a specific
sequence in a complex mixture by hybridization after blotting
onto filters (Northern blots), for which a method is described in
detail in Chapter 2.
2. Materials
2.1. Glyoxal Gel Method
1. 10x Electrophoresis buffer A: 100 mM sodium phosphate,
pH 6.7. The concentrated buffer should be sterilized by
autoclaving and diluted for use (to 10 mM) with sterile
water.
2. Dimethyl sulfoxide (DMSO).
3. Glyoxal. This can be obtained as a 40% (w/v) aqueous
solution or as a solid. Before use, remove oxidation prod-
ucts by treatment with a mixed-bed ion-exchange resin
RNA Gel Elecfrophoresis 3
(Amberlite MB-3, Biorad AG-501-X8, or equivalent). One
gram of resin is maintained in suspension in 10 mL of the
40% solution by slow magnetic stirring for I hat room tem-
perature. The deionized glyoxal solution is recovered by
filtration or by decanting after the resin beads have been
allowed to settle and may be stored in small amounts in
securely capped tubes at -70°C .
4. Electrophoresis grade agarose.
5. Denaturation mixture: DMSO/40% glyoxal/buffer A,
10/3/2 by vol.
6. Glycerol mix: glycerol/O.2% bromophenol blue in buffer
A, l/l by vol (seeN ote 4 in section 4).
2.2. FormaldehydeG el Method
1. 5x Electrophoresis buffer B: 0.2M sodium morpholino-
propane-sulfonate (MOPS), 50 mM EDTA, pH 7.0. Steril-
ize as for step 1 in section 2.1.
2. Formaldehyde: This is usually supplied as a 37% (w/v>
solution. The molecular weight of formaldehyde is 30.03,
so the concentration of the solution is 12.3M. The pH of
this solution should be greater than 4.0.
3. Formamide: This should be deionized as for glyoxal in
step 3 in section 2.1.
4. Denaturation mix: 50% (v/v) formamide, 2.2M formalde-
hyde, in 1 x electrophoresis buffer B (7).
5. Agarose bead loading buffer: Prepare by melting agarose
(0.2%) in 10 mM Tris-HCI, 20 mM EDTA, 10% (v/v>
glycerol, 0.2% bromophenol blue, pH 7.5. When the
mixture has solidified, it is forced several times through a
21-gage needle with a syringe to give a fine slurry. Store
at 4OC (seeN ote 4 in section 4).
2.3. Stainingo f RNA after Electrophoresis
1. 1% Toluidine blue-0 in 1% acetic acid.
2. Destaining solution: 1% acetic acid.
4 7%urstonP, erry, and Pollard
3. 1 &mL Ethidium bromide in distilled water.
4. O.lM Ammonium acetate.
5, 1 ug/mL Ethidium bromide in O.IM ammonium acetate.
3. Method
3.1. Glyoxal Gel Electrophoresis( see Fig. 1).
1. Cast 1.1% (w/v> agarose (seeNote 1 in section41 in electro-
phoresis buffer A as a 3-mm deep gel in an apparatus that
allows the gel to be submerged in buffer during electro-
phoresis and is equipped for recirculation of buffer be-
tween the electrode compartments. Amounts for this and
subsequent steps are given in Table 1 for typical gel appar-
atus of two different sizes.
2. Add RNA samples (seeN ote 2 in section 4) to 3 vol of denat-
uration mix in microfuge tubes and incubate capped at
50°C for 1 h. Cool to room temperature in ice/water.
3. Add 0.25 vol of glycerol mix to the denatured RNA sam-
ples. Use a positive displacement dispenser, since this is
viscous.
4. Fill the electrophoresis apparatus with buffer A so that the
gel is covered by a layer 3-5 mm deep.
5. Load the RNA samples and connect the power supply
such that the sample wells are at the cathode end of the gel
(seeN otes 3 and 4 in section 4).
6. Electrophoresis is performed with constant voltage to give
up to 4 V/cm (with respect to the distance between the
electrodes, not the length of the gel). Allow 10 min for
migration of RNA into the agarose and start the buffer
recirculation pump (seeT able 1). The bromophenol blue
marker dye migrates about 2.5 cm/h, and electrophoresis
should be stopped when the dye has migrated about 80%
of the distance from the sample wells to the end of the gel,
since tRNA migrates ahead of the dye.
RNA Gel Nectrophoresis 5
Fig. 1. Electrophoresis of glyoxal-denatured RNA, stained with toluid-
ine blue. Tracks contain (A) 10 pg total RNA from Aguricus bispouus,( B) 10
pg total RNA from Chlorellafusca (C), 1 pg tRNA from Escherichiu coli, (D) 3
pg I-phage DNA digested with the restriction enzyme Hind 111.
6 Thurston,P erry,a nd Pollard
Table 1
Composition of Gel and Sample Mixtures for Glyoxal Gels
of Two Common Sizes
Dimensions of the gel (mm) 100x64~3 200x200~3
Total volume of 300 mL 2200 mL
buffer A required
Agarose (in buffer A) 0.33 g in 30 mL 1.32 g in 120 mL
Denaturation mix:
DMSO 50 PL 100 FL
40% (w/v> glyoxal 15 PL 30 PL
Buffer A 10 PL 20 j.l.L
Glycerol mix:
Glycerol 10 PL 20 clr,
Buffer A 10 PL 20 PL
(+/- Bromophenol blue, seen ote 3 in section 4)
RNA in sterile deionized water 2 t.tL 4PL
(seen ote 2 in section 4)
Denaturation mix 6 ClL 12pL
Glycerol mix 2vL 4 w
Volume of sample loaded 10 j.tL 20 PL
Voltage 65V 120v
Buffer recirculation 200 mL/h 500 mL/h
Duration of electrophoresis
2h 3h
(approx.)
3.2. FormaldehydeG el Elecirophoresis
1. First melt the agarose in distilled water, and when it has
cooled to 6O”C, add 5x buffer B and formaldehyde. The
amounts for a minigel are: 0.3 g of agarose in 18.6 mL of
water, 6.0 mL of buffer (B), and 5.4 mL of formaldehyde
(this gives a 1% agarose gel, total volume, 30 mL; scale up
in proportion for larger gels).
RNA Gel Electrophoresis 7
2. Cast the gel (seeN ote 1 in section 4).
3. Place the gel in the electrophoresis apparatus and sub-
merge in lx buffer.
4. Add 4.5 PL of RNA solution (seeN ote 2 in section 4) to 2 PL
of 5x buffer B, 3.5 ~.I,Lfo rmaldehyde, and IO PL formamide.
Incubate in a capped tube for 15 min at 55OC. Cool in ice/
water.
5. Add 4 PL of agarose bead loading buffer and 2 PL of a 1
mg/ 1 mL ethidium bromide stock (seeN ote 5 in section 4)
to the denatured RNA sample and load into a sample well.
6. Electrophoresis is performed with constant current at 40V.
After 15 min start the buffer recirculation. Electrophoresis
is typically run overnight.
3.3. StainingR NAB andsa fterE lectrophoresis
1. Slide the gel from the plate (on which it was cast) into a tray
containing about 1 cm depth of 1% toluidine blue solution.
Stain for 15-60 min at room temperature, preferably on a
reciprocating shaker (10-30 strokes/min).
2. Drain off the staining solution and destain with several
changes of destain solution until the background of the gel
is completely clear (otherwise faintly stained bands will
not show up). Store the gel in destain solution.
3.4. Fluofescetn S taining
1. Glyoxal gels may be stained directly in aqueous ethidium
bromide, and RNA bands may be visualized on a UV
illuminator after about 30 min.
2. Formaldehyde gels must first be washed with distilled
water for 2 h, using four or five changes, in order to remove
the formaldehyde (seeN otes 5 and 6 in section 4).
3. After washing, soak the gel in O.lM ammonium acetate
twice for 1 h.
4. Stain for 1 h with ethidium bromide in ammonium acetate.
8 Thurston,P erry,a nd Pollard
5. Destain for 45 min in ammonium acetate and visualize on
a UV illuminator.
4. Notes
1. When preparing the agarose gel, it is essential to com-
pletely melt the agarose, which can be done successfully in
a microwaveoven, in a steamer, or with careful direct heat-
ing over a Bunsen burner. When the solution has gone
clear because the bulk of the agarose has dissolved, there
will still be a small proportion of swollen agarose beads
undissolved. These can be seen if the flask is held up to the
light. Continue heating until they have dissolved, since
they interfere with the RNA separation. At the same time
vigorous boiling must be avoided, since it can lead to
significant loss of water altering the agarose concentra-
tion. Allow the agarose to cool to about 50°C (not uncom-
fortably hot to touch) before pouring. If there are persis-
tent bubbles on the surface of the agarose after pouring,
they can be collapsed by briefly flaming the surface with a
Bunsen burner.
The protocols described give agarose concentrations
suitable for the separation of a wide range of molecular
weight species. When the marker bromophenol blue has
run 80% of the length of the gel, tRNA has not run off, and
16-18s ribosomal RNAs are approximately half way.
Other agarose concentrations may be more appropriate
for some specialized applications
2. Both of the methods described require relatively high con-
centrations of RNA because the samples are diluted with
denaturing reagents. Generally 5-50 pg of RNA are run on
the gel. If running total RNA either to assess integrity or
to transfer to a nitrocellulose filter for hybridization, 50 pg
might be a suitable amount. If separating mRNA for
transfer and hybridization, however, 5 pg would be suffi-
cient and would give superior resolution.
3. The loading buffers described all contain bromophenol
blue, but it is generally better to omit the tracker dye from
the samples and run the outside tracks of the gel with dye
but no sample. Most particularly do not include bromo-
phenol blue in molecular weight marker or sample tracks
if it is intended to visualize the RNA with ethidium bro-
mide: the dye can mask important RNA bands.
4. The incorporation of macerated agarose in sample mix-
tures improves the resolution (by reducing sample tailing)
in the formaldehyde system, as it does for electrophoresis
of DNA, but we have not been able to show that it has any
effect in the glyoxal system. In both systems great care
must be taken in sample application. The volume of
sample loaded must not fill the well above the surface of
the gel. This is possible because surface tension draws up
the agarose around the well-forming comb. If the sample
occupies that part of the well that is above the main body
of the gel surface, it streams at the gel-buffer interface. It
is equally important that the well-forming comb is clear of
the base plate so that the sample cannot leak out along the
underside of the gel.
5. For many purposes, ribosomal and tRNA markers are ade-
quate. The values for their molecular weights are shown
inTable 2. For blotting experiments (Chapter 21,r ibosom-
al markers run in flanking tracks are cut off and stained
with toluidine blue. This is convenient because the inten-
sity of staining does not diminish significantly for several
weeks, enabling direct comparison of the marker bands
with labeled bands visualized by autoradiography. Alter-
natively ethiduim bromide can be incorporated directly
into the sample and the rRNA bands visualized on a UV
transilluminator either on the gel or on the filter.
Use of DNA markers: Wild type A-phage DNA cut with
Hind 111t reated with glyoxal under the conditions de-
scribed for RNA migrate with the same relative mobility as
RNA (8). Under these conditions the DNA is singlestrand-
10 Thrston, Perry, and Pollard
Table 2
Molecular Weight of RNA and DNA Markers
Approximate
Ribosomal (and transfer) RNA 1O-6x MI no. of nucleotides
Mammalian
(mouse) 28s 1.70 4700
18s 0.71 1900
Chlorella 25s 1.18 3600
23s 1.00
18s 0.68 1900
16s 0.57 1800
5.8s 0.050 150
5s 0.037 110
tRNA 0.023 70
Escherichia coli 23s 1.07 2000
16s 0.56 1600
tRNA 0.025 75
h-Phage Hind III
restriction fragment DNA 7.13 23000
2.91 9400
2.05 6600
1.36 4380
0.71 2300
0.62 2000
0.174 560
0.039 125
ed, and consequently about 10 pg are required to give clear
staining. In the conditions of the formaldehyde gels, DNA
migrates more slowly than RNA of the same size (9).
Within the range of 0.5-7 million, RNA molecular weight
can be derived by adding 0.56 million to the molecular
weight of DNA, which has the same relative mobility.
6. If formaldehyde gels are processed for Northern blotting
(Chapter 2,Note l), they have been sufficiently depleted of