Analysis of RNA by Analytical Polyacrylamide Gel Electrophoresis

Transcription

1 CHAPTER SIXTEEN Analysis of RNA by Analytical Polyacrylamide Gel Electrophoresis Alexey Petrov, Albet Tsa, Joseph D. Puglisi 1 Stanford University School of Medicine, Stanford, CA, USA 1 Corresponding author: address: puglisi@stanford.edu Contents 1. Theory Equipment Materials Solutions & buffers Protocol Preparation Duration Step 1 Preparing the Gel Overview Duration Tip Tip Tip Tip Tip Step 2 Running the Gel Overview Duration Caution Step 3 Visualizing the RNA Overview Duration Caution Tip Tip Tip 311 References 313 Referenced Protocols in Methods Navigator 313 Methods in Enzymology, Volume 530 ISSN # 2013 Published by Elsevier Inc. 301

2 302 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,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 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

3 Analysis of RNA by Analytical Polyacrylamide Gel Electrophoresis 303 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 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, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. On a denaturing gel, RNA mobility is roughly inversely proportional to log 2 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

4 304 Alexey Petrov et al. Glass plates 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 (H 3 BO 3 ) EDTA Potassium hydroxide (KOH) Ammonium persulfate (APS) N,N,N 0,N 0 -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 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 with water. There is no need to adjust the ph of this solution

5 Analysis of RNA by Analytical Polyacrylamide Gel Electrophoresis 305 Native gel mix Component Final concentration Stock Amount TBE 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 Component Final concentration Stock Amount TBE 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 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 ml Glycerol 20% 100% 2 ml Bromophenol blue 0.05% 5 mg Xylene cyanol 0.05% 5 mg Add deionized water to 10 ml

6 306 Alexey Petrov et al. Running buffer Component Final concentration Stock Amount TBE 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 Duration Preparation Protocol About 2 h (þ time for RNA isolation) About 4 5 h See Fig for the flowchart of the complete protocol. Figure 16.1 Flowchart of the complete protocol, including preparation.

7 Analysis of RNA by Analytical Polyacrylamide Gel Electrophoresis STEP 1 PREPARING THE GEL 5.1. Overview Pour the gel. Prerun the gel (denaturing gel only) Duration 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 ( 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 V cm 1 for min to preheat the gel. Skip this step when running native gels 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 Tip Use large binder clips to clamp the gel plates and spacers together Tip Use RAIN-X Original Glass Treatment as an inexpensive alternative to other siliconizing agents.

8 308 Alexey Petrov et al Tip The aluminum plate helps ensure an even dissipation of heat, thus preventing overheating and uneven running across the gel Tip Vcm 1 is the total voltage divided by the distance between the gel rig electrodes in centimeter. See Fig for the flowchart of Step 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 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 V cm 1 ; run a native gel at V cm Use the mobility of the tracking dyes on the gel to determine when to stop running the gel. Acrylamide percentage Xylene cyanol co-migrates with Bromophenol blue co-migrates with 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, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

9 Figure 16.2 Flowchart of Step 1.

10 310 Alexey Petrov et al. Figure 16.3 Flowchart of Step Caution Switch off the power supply before loading the samples. See Fig for the flowchart of Step STEP 3 VISUALIZING THE RNA 7.1. Overview Stain or dry the gel Duration 3 h to stain the gel 1.5 h to dry the gelþovernight for autoradiography

11 Analysis of RNA by Analytical Polyacrylamide Gel Electrophoresis 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 Caution Switch off the power supply and disconnect the leads before disassembling the gel apparatus 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 Tip To speed up destaining, fold up a paper towel and submerge it in the water 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 for the flowchart of Step 3.

12 Figure 16.4 Flowchart of Step 3.

13 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, Sambrook, J., et al. (2001). Neutral polyacrylamide gel electrophoresis. Molecular Cloning. A Laboratory Manual. (pp ). 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.