RNA Purification by Preparative Polyacrylamide Gel ... · CHAPTER SEVENTEEN RNA Purification by Preparative Polyacrylamide Gel Electrophoresis Alexey Petrov, Tinghe Wu, Elisabetta

CHAPTER SEVENTEEN

RNA Purification by PreparativePolyacrylamide GelElectrophoresisAlexey Petrov, Tinghe Wu, Elisabetta Viani Puglisi, Joseph D. Puglisi1Stanford University School of Medicine, Stanford, CA, USA1Corresponding author: e-mail address: [email protected]

Contents

1. Theory 3162. Equipment 3183. Materials 318

3.1 Solutions & buffers 3194. Protocol 320

4.1 Preparation 3204.2 Duration 320

5. Step 1 Preparing the Gel 3215.1 Overview 3215.2 Duration 3215.3 Tip 3225.4 Tip 3225.5 Tip 3225.6 Tip 3225.7 Tip 322

6. Step 2 Running the Gel 3226.1 Overview 3226.2 Duration 3246.3 Caution 324

7. Step 3 Visualizing the RNA 3247.1 Overview 3247.2 Duration 3257.3 Caution 3257.4 Tip 3267.5 Tip 3267.6 Tip 326

8. Step 4A RNA Extraction Using the ‘Crush and Soak’ Method 3268.1 Overview 3268.2 Duration 327

Methods in Enzymology, Volume 530 # 2013 Published by Elsevier Inc.ISSN 0076-6879http://dx.doi.org/10.1016/B978-0-12-420037-1.00017-8

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8.3 Tip 3278.4 Tip 327

9. Step 4B RNA Extraction by Electroelution 3279.1 Overview 3279.2 Duration 3279.3 Tip 3299.4 Tip 329

References 330Referenced Protocols in Methods Navigator 330

Abstract

Preparative polyacrylamide gel electrophoresis (PAGE) is a powerful tool for purifyingRNA samples. Denaturing PAGE allows separation of nucleic acids that differ by a singlenucleotide in length. It is commonly used to separate and purify RNA species afterin vitro transcription, to purify naturally occurring RNA variants such as tRNAs, to removedegradation products, and to purify labeled RNA species. To preserve RNA integrity fol-lowing purification, RNA is usually visualized by UV shadowing or stained with ethidiumbromide or SYBR green dyes.

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,N 0-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–bisacrylamide ratio. The pore size

316 Alexey Petrov et al.

decreases with increasing acrylamide concentration, thus allowing the sep-

aration of smaller biomolecules (Holmes and Stellwagen, 1991). The ratio of

acrylamide to bisacrylamide affects the cross-linking frequency of the poly-

acrylamide mesh. Increase in the bisacrylamide concentration from 3.3%

(29:1 ratio of acrylamide to bisacrylamide) to 5% (19:1 ratio of acrylamide

to bisacrylamide) results in a decrease of the pore size, thus leading to a shift

in the separation range toward smaller RNA molecules. Further increase in

the concentration of bisacrylamide leads to an increase of the pore sizes due

to nonuniform chain cross-linking. A 19:1 ratio of acrylamide to bisa-

cylamide is commonly used for denaturing gel electrophoresis, while a

29:1 ratio of acrylamide to bisacylamide is used for native gel electrophoresis

of nucleic acids. The following table gives an approximate separation range

of RNAmolecules (in nucleotides) run on a native polyacrylamide gel (29:1

ratio of acrylamide to bisacrylamide). It is important to note that the sepa-

ration range for RNAmolecules run on a denaturing gel (19:1 ratio of acryl-

amide 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 poly-acrylamide gel electrophoresis. In: Molecular Cloning. A Laboratory Manual, pp. 5.42, 12.89. Cold SpringHarbor, NY: Cold Spring Harbor Laboratory Press.

On a denaturing gel, RNAmobility is roughly inversely proportional to

log2 of the size of the RNAmolecule. Thus, separation is better for the mol-

ecules at the smaller end of the separation range. While smaller percentage

(<10%) PAGE gels are excellent for separating large from very small RNAs,

higher percentage gels should be used for single-nucleotide resolution of

317RNA Purification by Preparative Polyacrylamide Gel Electrophoresis

larger (>60 nt) RNAs. We routinely use 20% PAGE for purification of

tRNAs (76 nt) at single-nucleotide resolution.

2. EQUIPMENT

PAGE gel apparatus

Power supply

UV illuminator (254 nm is preferable)

Nutator

Dry heat block

Electroelution apparatus

Glass plates

1.5 mm spacers

Gel comb

Micropipettors

Micropipettor tips

15-ml polypropylene tubes

Fluor-coated TLC plates (fluoresce in 254 nm UV light)

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,N 0,N 0-tetramethylethylenediamine (TEMED)

Formamide

Sodium dodecyl sulfate (SDS)

Bromophenol blue

Xylene cyanol

Ammonium acetate (NH4OAc)

Sodium acetate (NaOAc)

Ethanol


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! 40 ml

Acrylamide/bis-acrylamide (29:1) X% 40% (X/40)400 ml

Ammonium persulfate 0.08% 10% 3.2 ml

Add deionized water to 400 ml

Denaturing gel mix


TBE 1! 10! 40 ml

Acrylamide/bis-acrylamide (19:1) X% 40% (X/40)400 ml

Urea 6.5 M 10 M 260 ml

Ammonium persulfate 0.08% 10% 3.2 ml

Add deionized water to 400 ml

Step 2 2! Denaturing loading buffer


Formamide 95% 100% 9.5 ml

EDTA-KOH, pH 8.0 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 water to 10 ml


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

Add water to 10 ml

Running buffer


TBE 1! 10! 100 ml

Deionized water 900 ml

Step 4 Elution Solution


NH4OAc 500 mM 2 M 250 ml

EDTA-KOH, pH 8.0 1 mM 500 mM 2 ml

Add water to 1 l

70% Ethanol (v/v)


Ethanol 70% 96% 7.3 ml

Water 2.7 ml

4. PROTOCOL4.1. Preparation

Prepare stock solutions. Prepare the RNA sample to run on the gel.

4.2. Duration

Preparation About 2 h

Protocol About 6–10 h


See Fig. 17.1 for the flowchart of the complete protocol, including

preparation.

5. STEP 1 PREPARING THE GEL5.1. Overview

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

5.2. Duration1–1.5 h

1.1 Treat gel plates with a siliconizing agent.

1.2 Assemble the gel plates with 1.5 mm spacers.

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 160 ml of TEMED for every 400 ml of the gel mixture to start poly-

merization. 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.

Figure 17.1 Flowchart of the complete protocol, including preparation.


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 equilibrate

and preheat the gel. Skip this step for the native gels.

5.3. TipFirst, run an analytical gel to determine the optimal gel and running conditions (acryl-

amide percentage, ratio of acrylamide to bisacrylamide, run time, and run temperature.

See Analysis of RNA by analytical polyacrylamide gel electrophoresis.) that give the

desired resolution for your sample. Note the mobility of the RNA species relative to the

tracking dyes in a gel run under these conditions.

5.4. TipUse large binder clips to clamp the gel plates and spacers together. The gel plates can be

sealed using heavy-duty plastic tape such as Permacel P256 or 3 M Type 56 to help

prevent leaks.

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 uneven

running across the gel. The aluminum plates also help prevent the glass gel plates from

breaking at the high power required to keep the nucleic acids denatured while running

the gel.

5.7. TipV/cm is the total voltage divided by the distance between the gel rig electrodes in

centimeter.

See Fig. 17.2 for the flowchart of Step 1.

6. STEP 2 RUNNING THE GEL6.1. Overview

Prepare and load samples. Run the gel.


Figure 17.2 Flowchart of Step 1.

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 extensively with 1! TBE using micropipettor fitted

with a gel-loading tip. Load the samples into the wells.

2.4 Run a denaturing gel at 45–65 V cm#1; run a native gel 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.

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


7. STEP 3 VISUALIZING THE RNA7.1. Overview

The RNA molecules will be visualized by UV shadowing.


7.2. Duration30 min

3.1 Remove the gel plates from the gel running apparatus. Remove the

spacers. Use a metal spatula to pry open the top glass plate of the gel.

3.2 Cover the gel with plastic wrap. Carefully flip the gel plate so that the

side with the gel covered in plastic wrap is facing down. Remove the

glass plate. Wrap the gel in plastic wrap.

3.3 Place the gel on top of a fluorescent plate and shine UV light on it.

3.4 The RNA will be visible as dark spots on a brightly fluorescing

background.

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

apparatus.



7.4. TipFluorescent plates for UV shadowing are often referred to as ‘Fluor-coated TLC

plates’ and are available from several commercial suppliers.

7.5. TipCarry out the UV shadowing in a darkened environment, such as a darkroom, as this

will greatly improve band visibility.

7.6. TipThe bromophenol blue and xylene cyanol tracking dyes will be visible as dark spots as

well.


8. STEP 4A RNA EXTRACTION USING THE ‘CRUSH ANDSOAK’ METHOD

8.1. OverviewRNA will be extracted from the gel using the ‘crush and soak’ method (see

also Purification of DNA Oligos by Denaturing Polyacrylamide Gel

Electrophoresis (PAGE)).



8.2. Duration5 h

4A.1 Cut out the band of interest using a new, clean razor blade.

4A.2 Weigh a 50 -ml conical tube and note the weight of the empty tube.

Transfer the gel slice into the tube and crush the gel using a

disposable pipette.

4A.3 Weigh the tube containing the crushed gel slice and calculate the

weight of the gel. Add "2 volumes of Elution Solution (v/w) to

the gel.

4A.4 Incubate on a tube on a nutator at room temperature for 3 h.

4A.5 Centrifuge sample at 5000!g for 1 min. Collect the supernatant;

avoid picking up gel debris. Repeat the centrifugation two

more times.

4A.6 Add 0.1 volume of 3 M NaOAc, pH 5.2, and 3 volumes of ethanol.

Incubate at #20 $C for 1 h to precipitate the RNA.

4A.7 Collect the RNA by centrifugation at 15 000 rpm for 15 min at 4 $C.

Decant the supernatant and wash the pellet with 70% ethanol.

4A.8 Collect the RNA by centrifugation at 15 000 rpm for 15 min at 4 $C.

Decant the supernatant and air-dry the pellet.

4A.9 Resuspend the RNA pellet in water or a desired buffer.

8.3. TipCrush the gel thoroughly. Fine-crushing will decrease the elution time and increase

elution efficiency.

8.4. TipElution can be conducted in the cold room; however, the incubation time should be

extended to 6–9 h.

See Fig. 17.5 for the flowchart of Step 4A.

9. STEP 4B RNA EXTRACTION BY ELECTROELUTION9.1. Overview

RNA will be recovered from the gel by electroelution.

9.2. Duration3 h


4B.1 Assemble the electroelution apparatus according to the manufac-

turer’s instructions.

4B.2 Cut out the band of interest using a new, clean razor blade. Place the

gel slice in the electroelution chamber. Seal it well.

4B.3 Elute the RNA at 100 V for 2 h in 1! TBE, or according to the

manufacturer’s recommendations.

Figure 17.5 Flowchart of Step 4A.


4B.4 Recover the elutedRNA from the collection cup. Add 0.1 volume of

3 M NaOAc, pH 5.2, and 3 volumes of ethanol. Incubate at #20 $C

for 1 h to precipitate the RNA.

4B.5 Collect the RNA by centrifugation at 15 000 rpm for 15 min at 4 $C.

Decant the supernatant and wash the pellet with 70% ethanol.

4B.6 Collect the RNA by centrifugation at 15 000 rpm for 15 min at 4 $C.

Decant the supernatant and air-dry the pellet.

4B.7 Resuspend the RNA pellet in water or a desired buffer.

9.3. TipLeaking gaskets can be sealed with 1% agarose.

9.4. TipThe elution conditions can vary greatly depending on the electroelution apparatus, the

acrylamide percentage of the gel, the membranes, and the buffer used. A good starting

Figure 17.6 Flowchart of Step 4B.


point is to conduct the electrophoresis in 1! TBE at 100 V for 2 h. This should be

sufficient to elute "100 nucleotide long RNA molecules from a 4% polyacrylamide

gel. Conduct a test run to adjust the elution conditions.

See Fig 17.6 for the flowchart of Step 4B.

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 differentcrosslinker concentrations, added agarose and added linear polyacrylamide. Electrophore-sis, 12, 612–619.

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 HarborLaboratory 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 NAVIGATORAnalysis of RNA by analytical polyacrylamide gel electrophoresis.Purification of DNA Oligos by Denaturing Polyacrylamide Gel Electrophoresis (PAGE).


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RNA Purification by Preparative Polyacrylamide Gel ... · CHAPTER SEVENTEEN RNA Purification by Preparative Polyacrylamide Gel Electrophoresis Alexey Petrov, Tinghe Wu, Elisabetta