analysis of rna by analytical polyacrylamide gel ... · 2.2 heat the samples for the denaturing gel...

CHAPTER SIXTEEN

Analysis of RNA by AnalyticalPolyacrylamide GelElectrophoresisAlexey Petrov, Albet Tsa, Joseph D. Puglisi1Stanford University School of Medicine, Stanford, CA, USA1Corresponding author: e-mail address: [email protected]

Contents

1. Theory 3022. Equipment 3033. Materials 304

3.1 Solutions & buffers 3044. Protocol 306

4.1 Preparation 3064.2 Duration 306

5. Step 1 Preparing the Gel 3075.1 Overview 3075.2 Duration 3075.3 Tip 3075.4 Tip 3075.5 Tip 3075.6 Tip 3085.7 Tip 308

6. Step 2 Running the Gel 3086.1 Overview 3086.2 Duration 3086.3 Caution 310

7. Step 3 Visualizing the RNA 3107.1 Overview 3107.2 Duration 3107.3 Caution 3117.4 Tip 3117.5 Tip 3117.6 Tip 311

References 313Referenced Protocols in Methods Navigator 313

Methods in Enzymology, Volume 530 # 2013 Published by Elsevier Inc.ISSN 0076-6879http://dx.doi.org/10.1016/B978-0-12-420037-1.00016-6

301

http://dx.doi.org/10.1016/B978-0-12-420037-1.00016-6

Abstract

Polyacrylamide gel electrophoresis (PAGE) is a powerful tool for analyzing RNA samples.Denaturing PAGE provides information on the sample composition and structural integ-rity of the individual RNA species. Nondenaturing gel electrophoresis allows separationof the conformers and alternatively folded RNA species. It also can be used to resolveRNA protein complexes and to detect RNA complex formation by analyzing changes inthe electrophoretic mobility of the RNA. RNA can be visualized within gels by differentmethods depending on the nature of the detection reagent. RNA molecules can bestained with various dyes, including toluidine blue, SYBR green, and ethidium bromide.Radioactively labeled RNA molecules are visualized by autoradiography, and fluores-cently 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 mmare fragile and thus usually are not stained but rather are used for radiolabeled RNA. Thegels 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 prop-

erties 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 deter-

mines the separation properties of a polyacrylamide gel, wherein the rela-

tionship 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 acryl-

amide 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

302 Alexey Petrov et al.

acrylamide to bisacrylamide) results in a decrease of the pore size, thus lead-

ing 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 electro-

phoresis, while a 29:1 ratio of acrylamide to bisacrylamide is used for native

gel electrophoresis of nucleic acids. The following table gives an approxi-

mate separation range of RNA molecules (in nucleotides) run on a native

polyacrylamide gel (29:1 ratio of acrylamide to bisacrylamide). It is impor-

tant to note that the separation range for RNA molecules run on a denatur-

ing 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, RNAmobility is roughly inversely proportional to

log2 of the size of the RNA molecule. Thus, separation is better for mole-

cules 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

303Analysis of RNA by Analytical Polyacrylamide Gel Electrophoresis

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 Stock Amount

Tris base 890 mM 108 g

EDTA, pH 8.0 20 mM 0.5 M 40 ml

Boric acid 890 mM 55 g

Dissolve tris and boric acid in "750 ml of deionized water. Add EDTA. Adjust final volume to 1 l withwater. There is no need to adjust the pH of this solution


Native gel mix


TBE 1! 10! 10 ml

Acrylamide/bis-acrylamide (29:1) X% 40% (X/40)100 ml

Ammonium persulfate 0.08% 10% 800 ml

Add deionized water to 100 ml

Denaturing gel mix


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


2! Denaturing loading buffer


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


5! Nondenaturing loading buffer


TBE 5! 10! 5 ml

Glycerol 20% 100% 2 ml

Bromophenol blue 0.05% 5 mg

Xylene cyanol 0.05% 5 mg



Running buffer


TBE 1! 10! 100 ml

Deionized water 900 ml

Step 3 Staining solution

Component Final concentration Amount

Toluidine blue 0.1% 1 g

Acetic acid 10% 100 ml

Deionized water 900 ml

4. PROTOCOL4.1. 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.


5. STEP 1 PREPARING THE GEL5.1. Overview

Pour the gel. Prerun the gel (denaturing gel only).

5.2. Duration1–1.5 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 thewells with

1! TBE using a micropipettor fitted with a gel-loading tip.

1.6 For denaturing gels larger than 20!20 cm, clamp an aluminum plate to

the front side of the gel plate.

1.7 Prerun denaturing gels at 45–65 V cm$1 for 30–60 min to preheat the

gel. Skip this step when running native gels.

5.3. TipIn 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. TipUse large binder clips to clamp the gel plates and spacers together.

5.5. TipUse RAIN-X® Original Glass Treatment as an inexpensive alternative to other

siliconizing agents.


5.6. TipThe aluminum plate helps ensure an even dissipation of heat, thus preventing over-

heating and uneven running across the gel.

5.7. TipV cm$1 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 GEL6.1. Overview

Prepare and load samples (see labeling methods on RNARadiolabeling and

Fluorescently Labeling Synthetic RNAs). Run the gel.

6.2. DurationVariable, 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! non-

denaturing 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 gel-

loading tip. Load the samples into the wells.

2.4 Runadenaturinggel at 45–65 V cm$1; runanativegel at 10–25 V cm$1.

2.5 Use the mobility of the tracking dyes on the gel to determine when to

stop running the gel.

Acrylamide percentage Xylene cyanol co-migrates withBromophenol blueco-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

Numbers represent approximate RNA size in nucleotides. From Sambrook J, et al. (2001) Neutral poly-acrylamide gel electrophoresis. In: Molecular Cloning. A Laboratory Manual, pp. 5.42, 12.89. Cold SpringHarbor, NY: Cold Spring Harbor Laboratory Press.


Figure 16.2 Flowchart of Step 1.

6.3. CautionSwitch off the power supply before loading the samples.


7. STEP 3 VISUALIZING THE RNA7.1. Overview

Stain or dry the gel.

7.2. Duration3 h to stain the gel

1.5 h to dry the gelþovernight for autoradiography



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. CautionSwitch off the power supply and disconnect the leads before disassembling the gel

apparatus.

7.4. TipGenerally, 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. TipTo speed up destaining, fold up a paper towel and submerge it in the water.

7.6. TipWet the surface of the gel with a small amount of water so that it will stick better to the

Whatman paper.



REFERENCESReferenced LiteratureHolmes, 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 crosslinkerconcentrations, 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 SpringHarbor Laboratory Press.

Stellwagen, N. C. (2009). Electrophoresis of DNA in agarose gels, polyacrylamide gels and infree solution. Electrophoresis, 30(supplement 1), S188–S195.

REFERENCED PROTOCOLS IN METHODS NAVIGATORRNA Radiolabeling.Fluorescently Labeling Synthetic RNAs.


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analysis of rna by analytical polyacrylamide gel ... · 2.2 heat the samples for the denaturing gel...

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