new nucleic acid techniques

Chapter 1 Electrophoresis of RNA Denatured with Glyoxal or Formaldehyde Christopher F. Thurston, Caroline R. Perry, and Jeffrey W. Pollard 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, Perry, and 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 (see Note 4 in section 4). 2.2. Formaldehyde Gel Method 1. 2. 3. 4. 5. 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. 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. Formamide: This should be deionized as for glyoxal in step 3 in section 2.1. Denaturation mix: 50% (v/v) formamide, 2.2M formalde- hyde, in 1 x electrophoresis buffer B (7). 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 (see Note 4 in section 4). 2.3. Staining of RNA after Electrophoresis 1. 1% Toluidine blue-0 in 1% acetic acid. 2. Destaining solution: 1% acetic acid. 4 7%urston, Perry, 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. 1. 2. 3. 4. 5. 6. Glyoxal Gel Electrophoresis (see Fig. 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. Add RNA samples (see Note 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. Add 0.25 vol of glycerol mix to the denatured RNA sam- ples. Use a positive displacement dispenser, since this is viscous. Fill the electrophoresis apparatus with buffer A so that the gel is covered by a layer 3-5 mm deep. Load the RNA samples and connect the power supply such that the sample wells are at the cathode end of the gel (see Notes 3 and 4 in section 4). 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 (see Table 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, Perry, and 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 buffer A required Agarose (in buffer A) 300 mL 2200 mL 0.33 g in 30 mL 1.32 g in 120 mL Denaturation mix: DMSO 40% (w/v> glyoxal Buffer A 50 PL 100 FL 15 PL 30 PL 10 PL 20 j.l.L Glycerol mix: Glycerol 10 PL 20 clr, Buffer A 10 PL 20 PL (+/- Bromophenol blue, see note 3 in section 4) RNA in sterile deionized water 2 t.tL 4PL (see note 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 (approx.) 2h 3h 3.2. Formaldehyde Gel 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. 3. 4. 5. 6. Cast the gel (see Note 1 in section 4). Place the gel in the electrophoresis apparatus and sub- merge in lx buffer. Add 4.5 PL of RNA solution (see Note 2 in section 4) to 2 PL of 5x buffer B, 3.5 ~.I,L formaldehyde, and IO PL formamide. Incubate in a capped tube for 15 min at 55OC. Cool in ice/ water. Add 4 PL of agarose bead loading buffer and 2 PL of a 1 mg/ 1 mL ethidium bromide stock (see Note 5 in section 4) to the denatured RNA sample and load into a sample well. Electrophoresis is performed with constant current at 40V. After 15 min start the buffer recirculation. Electrophoresis is typically run overnight. 3.3. Staining RNA Bands after Electrophoresis 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. Fluofescen t Staining 1. 2. 3. 4. Glyoxal gels may be stained directly in aqueous ethidium bromide, and RNA bands may be visualized on a UV illuminator after about 30 min. Formaldehyde gels must first be washed with distilled water for 2 h, using four or five changes, in order to remove the formaldehyde (see Notes 5 and 6 in section 4). After washing, soak the gel in O.lM ammonium acetate twice for 1 h. Stain for 1 h with ethidium bromide in ammonium acetate. 8 Thurston, Perry, and 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. 4. 5. 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. 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. 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, ribosom- 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 111 treated 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-6 x MI no. of nucleotides Mammalian (mouse) 28s 18s 1.70 0.71 4700 1900 Chlorella 25s 1.18 23s 1.00 18s 0.68 16s 0.57 5.8s 0.050 5s 0.037 tRNA 0.023 3600 1900 1800 150 110 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 [...]... to the wash in 20x SSC References 2 2 3 4 5 6 7 8 9 Loening, V E (1967) The fractionation of high-molecular weight ribonucleic acid by polyacrylamide-gel electrophoresis Biochem 1, 102,251-257 McMaster, G K and Carmichael, G G (1977) Analysis of single and double-stranded nucleic acids on polyacrylamide gels by using glyoxal and acridine orange Proc N&Z Ad Sci USA 74,4835-4838 Lehrach, H., Diamond,... under denaturing conditions, a critical re-examination Biochemistry 16, 4743-4751 Staynov, D Z., Pinder, J C., and Gratzer, W B (1972) Molecular weight determination of nucleic acids by gel electrophoresis in nonaqueous solution Nature New Bid 235,108110 Reijnders, L., Sloof, P.,Sival, J., and Borst,P (1973) Gel electrophoresis of RNA under denaturing conditions Biochem Biophys Actu 324,320333 Bailey,... for agarose gel electrophoresis Anal Biochem 70, 75-80 Maniatis, T., Fritsch, E F., and Sambrook, J (1982) Molecular cloning A Laboratory Manual Cold Spring Harbor, New York Carmichael, G G and McMaster, G K (1980) The analysis of nucleic acids in gels using glyoxal and acridine orange Mefh EnzymoZ 65, 380-391 Wicks, R J (1986) RNA molecular weight determination by agarose gel electrophoresis using formaldehyde... filter Specific sequences are then detected by hybridization The RNA species are both immobilized on the filter and denatured so that when the filter is immersed in a solution containing a labeled nucleic acid probe, the probe binds to RNA of complementary base sequence (hybridizes), specifically labeling the position on the filter that this RNA sequence has been blotted to from the gel This defines... nitrocel13 14 Pollard,Perry, and Thufston lulose or nylon filters and its subsequent hybridization A typical result is shown in Fig 1 In this and many other procedures it is necessary to label the nucleic acid being used as a hybridization probe, and we therefore include a protocol for nick translation of DNA (3) It should be noted that there are now a number of alternatives to this procedure (seeNote... pg/mL poly A; 0.05Msodium phosphate, pH 7.0 16 Pollard, Perry,and Thurston 10 Hybridization mix: all the components of the prehybridization mix, except the poly A, but in addition, labeled probe nucleic acid (typically 200 ng in 600 PL) 11 Wash buffers: (A) 2x SSC,0.1% SDS, 0.05% sodium pyrophosphate (B) lx SSC, 0.1% SDS, 0.05% sodium pyrophosphate (C) 0.1x SSC, 0.1% SDS, 0.05% sodium pyrophosphate... No 1 paper for 30-60 min 14 Bake the dry membrane filter at 80°C for 2 h After baking, the membrane filter may be stored desiccated for many months prior to hybridization Baking fixes the adsorbed nucleic acid to the membrane filter 3.2 labeling of ProbeDNA by Nick Translation 1 2 3 4 5 6 7 Make up the following reaction mixture in a sterile microfuge tube: 2.5 PL of 10x nick-translation buffer, 2.5... highly labeled probes (5) and Bogorad’s procedure (6) for endlabeling RNA with T4 polynucleotide kinase is also very useful The purpose of prehybridization is to minimize nonspecific binding of probe nucleic acid to the membrane filter This step can be run overnight for convenience The inclusion of 30% formamide allows the hybridization to be carried out at 37-42”C In its absence the temperature would... Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose Pm Nufl Acud Sci USA 77, 5201-5205 Rigby, P W J., Dieckmann, M., Rhodes, C., and Berg, P (1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase1 J Mol Biol 113,237-251 Technical Bulletin 80/3 (1980) Labelling of DNA with 32p by nick translation Amersham International,... Rebagliati, M R., Maniatis, T.,Zinn,K.,and Green, M R (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter Nucleic Acid Res 12,7035-7056 Bogorad, L., Gubbins, E J., Krebbers, E., Larrinva, I M., Mulligan, B., Muskavitch, K M T., Orr, E A., Rodermal, S R., Schantz, R., Steinmetz, A A., De Vos, G., and Ye, Y K (1983) . weight ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem. 1, 102,251-257. McMaster, G. K. and Carmichael, G. G. (1977) Analysis of single and double-stranded nucleic acids on polyacrylamide. and Gratzer, W. B. (1972) Molecular weight determination of nucleic acids by gel electrophoresis in non- aqueous solution. Nature New Bid. 235,108110. Reijnders, L., Sloof, P.,Sival, J., and. Molecular cloning. A Laboratory Manual Cold Spring Harbor, New York. Carmichael, G. G. and McMaster, G. K. (1980) The analysis of nucleic acids in gels using glyoxal and acridine orange. Mefh.