CHAPTER SIXTEEN
Analysis of RNA by Analytical
Polyacrylamide Gel
Electrophoresis
Alexey Petrov, Albet Tsa, Joseph D. Puglisi1
Stanford University School of Medicine, Stanford, CA, USA
1
Corresponding author: e-mail address: puglisi@stanford.edu
Contents
1. Theory
2. Equipment
3. Materials
3.1 Solutions & buffers
4. Protocol
4.1 Preparation
4.2 Duration
5. Step 1 Preparing the Gel
5.1 Overview
5.2 Duration
5.3 Tip
5.4 Tip
5.5 Tip
5.6 Tip
5.7 Tip
6. Step 2 Running the Gel
6.1 Overview
6.2 Duration
6.3 Caution
7. Step 3 Visualizing the RNA
7.1 Overview
7.2 Duration
7.3 Caution
7.4 Tip
7.5 Tip
7.6 Tip
References
Referenced Protocols in Methods Navigator
Methods in Enzymology, Volume 530
ISSN 0076-6879
http://dx.doi.org/10.1016/B978-0-12-420037-1.00016-6
302
303
304
304
306
306
306
307
307
307
307
307
307
308
308
308
308
308
310
310
310
310
311
311
311
311
313
313
#
2013 Published by Elsevier Inc.
301
302
Alexey Petrov et al.
Abstract
Polyacrylamide gel electrophoresis (PAGE) is a powerful tool for analyzing RNA samples.
Denaturing PAGE provides information on the sample composition and structural integrity of the individual RNA species. Nondenaturing gel electrophoresis allows separation
of the conformers and alternatively folded RNA species. It also can be used to resolve
RNA protein complexes and to detect RNA complex formation by analyzing changes in
the electrophoretic mobility of the RNA. RNA can be visualized within gels by different
methods depending on the nature of the detection reagent. RNA molecules can be
stained with various dyes, including toluidine blue, SYBR green, and ethidium bromide.
Radioactively labeled RNA molecules are visualized by autoradiography, and fluorescently labeled RNA molecules can be observed with a fluorescence scanner. Generally,
gels between 0.4 and 1.5 mm thick are used for analytical PAGE. Gels thinner than 1 mm
are fragile and thus usually are not stained but rather are used for radiolabeled RNA. The
gels are dried and the radiolabeled RNA is visualized by autoradiography.
1. THEORY
Charged biomolecules migrate through electric fields with velocities
proportional to their charge and the strength of the electric field. The nature
of the gel matrix and the buffer composition determine the separation properties of the gel. Polyacrylamide meshes are commonly used to separate
nucleic acids. In denaturing polyacrylamide gels, the separation occurs
largely according to the size of the molecule, whereas in nondenaturing gels,
nucleic acid mobility is determined by both the size and conformation
(Stellwagen, 2009). Polyacrylamide gels are formed by the polymerization
of acrylamide in the presence of a cross-linking reagent, which is commonly
N,N0 -methylenebisacrylamide (referred to as bisacrylamide). This results in a
mesh-like network where long acrylamide fibers are cross-linked via
bisacrylamide bridges. The size-sieving effect is the main factor that determines the separation properties of a polyacrylamide gel, wherein the relationship between the size of the pores and the size of the molecule
determines the relative mobility of RNA through a polyacrylamide gel.
The apparent pore size is mainly affected by two parameters: the total acrylamide concentration and the acrylamide to bisacrylamide ratio. The pore
size decreases with increasing acrylamide concentration, thus allowing the
separation of smaller biomolecules (Holmes and Stellwagen, 1991). The
ratio of acrylamide to bisacrylamide affects the cross-linking frequency of
the polyacrylamide mesh. An increase in the bisacrylamide concentration
from 3.3% (29:1 ratio of acrylamide to bisacrylamide) to 5% (19:1 ratio of
303
Analysis of RNA by Analytical Polyacrylamide Gel Electrophoresis
acrylamide to bisacrylamide) results in a decrease of the pore size, thus leading to a shift in the separation range toward smaller RNA molecules.
A further increase in the concentration of bisacrylamide leads to an increase
of the pore sizes because of nonuniform chain cross-linking. A 19:1 ratio of
acrylamide to bisacrylamide is commonly used for denaturing gel electrophoresis, while a 29:1 ratio of acrylamide to bisacrylamide is used for native
gel electrophoresis of nucleic acids. The following table gives an approximate separation range of RNA molecules (in nucleotides) run on a native
polyacrylamide gel (29:1 ratio of acrylamide to bisacrylamide). It is important to note that the separation range for RNA molecules run on a denaturing gel (19:1 ratio of acrylamide to bisacrylamide) is approximately half that
for RNA molecules run on a native gel.
Acrylamide percentage
Separation range
3.5
500–2000
5.0
80–500
8.0
60–400
12.0
40–200
15.0
25–150
20.0
6–100
Numbers represent approximate RNA size in nucleotides. From
Sambrook J, et al. (2001) Neutral polyacrylamide gel electrophoresis.
In: Molecular Cloning. A Laboratory Manual, pp. 5.42, 12.89. Cold Spring
Harbor, NY: Cold Spring Harbor Laboratory Press.
On a denaturing gel, RNA mobility is roughly inversely proportional to
log2 of the size of the RNA molecule. Thus, separation is better for molecules at the smaller end of the separation range. For example, while both 6%
and 12% denaturing gels could be used to separate RNA species between
70 and 75 nucleotides (see table above), an 8% gel offers better resolution
at this size range.
2. EQUIPMENT
PAGE gel apparatus
Power supply
Platform rotator
Vacuum gel dryer
304
Alexey Petrov et al.
Glass plates
0.4–1.5 mm spacers
Gel comb
Staining dish
Micropipettors
Micropipettor tips
15-ml polypropylene tubes
3. MATERIALS
40% acrylamide/bisacrylamide (19:1)
40% acrylamide/bisacrylamide (29:1)
Urea
Tris base
Boric acid (H3BO3)
EDTA
Potassium hydroxide (KOH)
Ammonium persulfate (APS)
N,N,N0 ,N0 -tetramethylethylenediamine (TEMED)
Formamide
Sodium dodecyl sulfate (SDS)
Bromophenol blue
Xylene cyanol
Acetic acid
Toluidine blue
3.1. Solutions & buffers
Step 1 10 TBE
Component
Final concentration
Tris base
890 mM
EDTA, pH 8.0
20 mM
Boric acid
890 mM
Stock
Amount
108 g
0.5 M
40 ml
55 g
Dissolve tris and boric acid in 750 ml of deionized water. Add EDTA. Adjust final volume to 1 l with
water. There is no need to adjust the pH of this solution
305
Analysis of RNA by Analytical Polyacrylamide Gel Electrophoresis
Native gel mix
Component
Final concentration
Stock
Amount
TBE
1
10 10 ml
Acrylamide/bis-acrylamide (29:1)
X%
40%
(X/40)100 ml
Ammonium persulfate
0.08%
10%
800 ml
Component
Final concentration
Stock
Amount
TBE
1
10 10 ml
Acrylamide/bis-acrylamide (19:1)
X%
40%
(X/40)100 ml
Urea
6.5 M
10 M
65 ml
Ammonium persulfate
0.08%
10%
800 ml
Add deionized water to 100 ml
Denaturing gel mix
Add deionized water to 100 ml
2 Denaturing loading buffer
Component
Final concentration
Stock
Amount
Formamide
95%
100%
9.5 ml
EDTA
18 mM
500 mM
360 ml
SDS
0.025%
10%
25 ml
Bromophenol blue
0.05%
5 mg
Xylene cyanol
0.05%
5 mg
Add deionized water to 10 ml
5 Nondenaturing loading buffer
Component
Final concentration
Stock
Amount
TBE
5
10 5 ml
Glycerol
20%
100%
2 ml
Bromophenol blue
0.05%
5 mg
Xylene cyanol
0.05%
5 mg
Add deionized water to 10 ml
306
Alexey Petrov et al.
Running buffer
Component
Final concentration
Stock
Amount
TBE
1
10 100 ml
Deionized water
900 ml
Step 3 Staining solution
Component
Final concentration
Amount
Toluidine blue
0.1%
1g
Acetic acid
10%
100 ml
Deionized water
900 ml
4. PROTOCOL
4.1. Preparation
Prepare stock solutions. Isolate or obtain RNA to run on the gel.
4.2. Duration
Preparation
About 2 h (þ time for RNA isolation)
Protocol
About 4–5 h
See Fig. 16.1 for the flowchart of the complete protocol.
Figure 16.1 Flowchart of the complete protocol, including preparation.
Analysis of RNA by Analytical Polyacrylamide Gel Electrophoresis
307
5. STEP 1 PREPARING THE GEL
5.1. Overview
Pour the gel. Prerun the gel (denaturing gel only).
5.2. Duration
1–1.5 h
1.1 For gels 0.4 mm in thickness, treat the gel plates with a siliconizing
agent.
1.2 Assemble the gel plates with spacers of the desired thickness
(0.4–1.5 mm).
1.3 Prepare the appropriate gel mixture (for native or denaturing gels). The
percentage of acrylamide depends on the sizes of the RNA molecules
you wish to resolve.
1.4 Add 40 ml of TEMED for every 100 ml of the gel mixture to start
polymerization. Quickly mix the solution (without introducing air
bubbles) and pour the gel. Insert the desired comb and allow the gel
to polymerize.
1.5 Mount the gel plates onto the gel running apparatus. Add 1 TBE to both
the upper and lower reservoirs. Remove the comb and rinse the wells with
1 TBE using a micropipettor fitted with a gel-loading tip.
1.6 For denaturing gels larger than 2020 cm, clamp an aluminum plate to
the front side of the gel plate.
1.7 Prerun denaturing gels at 45–65 V cm1 for 30–60 min to preheat the
gel. Skip this step when running native gels.
5.3. Tip
In general, gels between 0.4 and 1.5 mm in thickness are used for analytical PAGE.
Gels thinner than 1 mm are fragile and thus usually are not stained but instead are
dried and used to detect radiolabeled samples. If you are staining the gel, pour it using
thicker spacers.
5.4. Tip
Use large binder clips to clamp the gel plates and spacers together.
5.5. Tip
Use RAIN-X® Original Glass Treatment as an inexpensive alternative to other
siliconizing agents.
308
Alexey Petrov et al.
5.6. Tip
The aluminum plate helps ensure an even dissipation of heat, thus preventing overheating and uneven running across the gel.
5.7. Tip
V cm1 is the total voltage divided by the distance between the gel rig electrodes in
centimeter.
See Fig. 16.2 for the flowchart of Step 1.
6. STEP 2 RUNNING THE GEL
6.1. Overview
Prepare and load samples (see labeling methods on RNA Radiolabeling and
Fluorescently Labeling Synthetic RNAs). Run the gel.
6.2. Duration
Variable, depends on the gel size
2.1 Mix the RNA sample with the appropriate loading buffer. If running a
denaturing gel, add equal volumes RNA sample and 2 denaturing
loading buffer. If running a native gel, add 1 volume of 5 nondenaturing loading buffer to 4 volumes of RNA sample.
2.2 Heat the samples for the denaturing gel at 94 C for 5 min.
2.3 Rinse the wells with 1 TBE using a micropipettor fitted with a gelloading tip. Load the samples into the wells.
2.4 Run a denaturing gel at 45–65 V cm1; run a native gel at 10–25 V cm1.
2.5 Use the mobility of the tracking dyes on the gel to determine when to
stop running the gel.
Xylene cyanol co-migrates with
Bromophenol blue
co-migrates with
3.5
460
100
5.0
260
65
8.0
160
45
12.0
70
20
15.0
60
15
20.0
45
12
Acrylamide percentage
Numbers represent approximate RNA size in nucleotides. From Sambrook J, et al. (2001) Neutral polyacrylamide gel electrophoresis. In: Molecular Cloning. A Laboratory Manual, pp. 5.42, 12.89. Cold Spring
Harbor, NY: Cold Spring Harbor Laboratory Press.
Figure 16.2 Flowchart of Step 1.
310
Alexey Petrov et al.
Figure 16.3 Flowchart of Step 2.
6.3. Caution
Switch off the power supply before loading the samples.
See Fig. 16.3 for the flowchart of Step 2.
7. STEP 3 VISUALIZING THE RNA
7.1. Overview
Stain or dry the gel.
7.2. Duration
3 h to stain the gel
1.5 h to dry the gelþovernight for autoradiography
Analysis of RNA by Analytical Polyacrylamide Gel Electrophoresis
311
3.1 Remove the gel plates from the gel running apparatus.
3.2 Remove the spacers. Use a metal spatula to pry open the top glass plate
without tearing the gel.
3.3 To stain a gel, transfer it into a staining dish slightly larger than the gel.
Add enough staining solution to cover the gel.
3.4 Incubate on a platform rotator for 1 h.
3.5 Decant the staining solution. Destain the gel in water, changing the
water every 30 min. The RNA will appear as blue-colored bands.
3.6 To dry a gel, place a sheet of Whatman 3MM chromatography paper
on top of the gel. Gently press the paper onto the gel surface to ensure a
uniform contact between the gel and the paper.
3.7 Lift a corner of the paper with the gel attached, carefully peeling the gel
from the glass plate.
3.8 Cover the gel with plastic wrap and dry it for 1 h at 80 C using a
vacuum gel dryer.
3.9 Visualize RNA by autoradiography.
7.3. Caution
Switch off the power supply and disconnect the leads before disassembling the gel
apparatus.
7.4. Tip
Generally, gels thicker than 1 mm can be stained. Thinner gels should be transferred
to a piece of Whatman 3MM chromatography paper that is used as a support media,
and then dried.
7.5. Tip
To speed up destaining, fold up a paper towel and submerge it in the water.
7.6. Tip
Wet the surface of the gel with a small amount of water so that it will stick better to the
Whatman paper.
See Fig. 16.4 for the flowchart of Step 3.
Figure 16.4 Flowchart of Step 3.
Analysis of RNA by Analytical Polyacrylamide Gel Electrophoresis
313
REFERENCES
Referenced Literature
Holmes, D. L., & Stellwagen, N. C. (1991). Estimation of polyacrylamide gel pore size from
Fergwson plots of linear DNA fragments 11. Comparison of gels with different crosslinker
concentrations, added agarose and added linear polyacrylamide. Electrophoresis, 12,
612–619.
Sambrook, J., et al. (2001). Neutral polyacrylamide gel electrophoresis. Molecular Cloning.
A Laboratory Manual. (pp. 5.42–12.89). Cold Spring Harbor, NY: Cold Spring
Harbor Laboratory Press.
Stellwagen, N. C. (2009). Electrophoresis of DNA in agarose gels, polyacrylamide gels and in
free solution. Electrophoresis, 30(supplement 1), S188–S195.
REFERENCED PROTOCOLS IN METHODS NAVIGATOR
RNA Radiolabeling.
Fluorescently Labeling Synthetic RNAs.