[ 17] TISSUE RNA AS SOURCE OF ION CHANNELS 297

Methods for cloning PCR products in transcription vectors using linkers or adaptors can be found in Ref. 19. A more versatile system, namely, the TA cloning kit from Invitrogen (San Diego, CA) has recently been introduced which may be an easier alternative to methods using linkers and adapters. This system utilizes the fact that thermophilic en- zymes used for PCR amplification have inherent terminal transferase ac- tivity, resulting in the generation of PCR products containing 3' overhangs composed of a single deoxyadenylate residue which can be directly ligated to a vector containing single 5'-deoxythymidylate overhangs. Alterna- tively, oligonucleotide primers containing unique restriction sites can be used for amplification of target sequences, 22 eliminating the ligation step with linkers or adapters.

Acknowledgments

The authors wish to thank Drs. Terry Snutch, Jeff Hoger, Henry l_ester, and Norman Davidson for the methylmercury gel electrophoresis and electroelution procedure. A.L.G. is a Lucille P. Markey Scholar. Work in the authors' laboratories is supported by grants from the U.S. National Institutes of Health (NS-25928, NS-27341, and NS-26729), and Muscular Dystrophy Association, the Lueille P. Markey Charitable Trust, and the March of Dimes Basil O'Connor Starter Scholar Program.

~2 M. A. Frohman, M. K. Dush, and G. R. Martin, Proc. NatL Acad. Sci. U.S.A. 86, 8998 (1988).

[ 17] Tissue R N A as Source of Ion Channels and Receptors

By TERRY P. SNUTCH and GAIL MANDEL

Introduction

The many attributes of the Xenopus oocyte expression system have already been outlined by Soreq and Seidman ([14] in this volume). In addition to healthy oocytes, the successful expression of exogenous excit- ability proteins requires intact, biologically active RNA. The RNA may be synthesized in vitro from a clone (see [16] and [18], this volume), or cellular RNA may be purified from a cell line or tissue known to express the ion channel or receptor of interest. If the molecule under study is a

Copyright © 1992 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL 207 All fights ofreproduction in any form reserved.

298 EXPRESSmN OF ION CnANNELS [ 17]

multisubunit complex, then coexpression of the complete complement of mRNAs is likely to be required to reconstitute all functional propertiesJ ,2

Many excitable proteins are encoded by large RNAs [> 5 kilobases (kb)], and special care must be taken to avoid degradation of the RNA. In addition, the health of the oocytes is impaired when they are microinjected with RNA samples contaminated by salts and detergents which were not removed during purification. This chapter outlines the conditions required to isolate high molecular weight cellular RNA suitable for exogenous expression. We also describe several protocols (gel electrophoresis, North- ern blot analysis, RNase protection) useful for the initial characterization of purified RNA.

Enzymatic and Chemical Considerations

RNA is highly susceptible to both enzymatic and chemical degradation. During the initial homogenization ribonuclease is likely to be introduced by lysis of cells and organelles. In the later stages of purification ribonucle- ases are most likely to be introduced from exogenous sources. Chemical degradation of RNA can occur at all stages of RNA purification, handling, and storage. Because the initial homogenization is carried out in the pres- ence of strong ribonuclease inhibitors, there is little concern for further precautions at this stage. However, once the partially purified RNA is removed from these strong denaturing agents, any further introduction of ribonuclease is detrimental to success. The most serious problems are ribonucleases which can contaminate glassware, solutions, equipment, and laboratory workers. A number of precautions can be taken to minimize ribonuclease contamination3:

1. Use only the highest quality reagents, for example, ultrapure molec- ular biology grade.

2. Laboratory glassware should be baked overnight at 180 °. Alterna- tively, glassware can be treated by soaking with 0.1% diethyl pyrocarbonate (DEPC) for 60 min, rinsing several times with distilled water, and then autoclaving for 30 min.

3. Laboratory utensils for making solutions (e.g., stir bars and weighing spatulas) should be routinely baked overnight at 180°.

4. Ribonucleases are derived from living organisms, and they are

M. M. White, K. Mixter-Mayne, H. A. I.ester, and N. Davidson, Proc. Natl. Acad. Sci. U.S.A. 82, 4852 (1985).

2 D. B. Pritchett, H. Sontheimer, B. D. Shivers, S. Ymer, H. Kettenmann, P. R. Schofield, and P. H. Seeburg, Nature (London) 338, 582 (1989).

a D. D. Blumberg, this series, Vol. 152, p. 20.

[ 17] xissu~ RNA As SOURCE OF ION CHANNELS 299

found on the skin and in the sweat of humans. To prevent introducing ribonuclease contamination, always wear disposable gloves when handling RNA samples and making stock solutions.

5. When ribonuclease-free glassware is not available, use disposable, sterile plasticware.

6. The pH meter is a likely source of ribonuclease and should not come into direct contact with solutions which are to be used after the initial homogenization of the tissue or cell line.

7. Keep a separate set of pipetting devices and an agarose gel system (combs, spacers, and gel box) for RNA use only.

8. We have not had problems with ribonuclease contamination when all laboratory glassware is routinely baked and when using autoclaved glass-distilled water and molecular biology grade reagents. However, if ribonuclease contamination is a problem, then solutions should be treated with DEPC. Using a stirrer, dissolve DEPC into the solution to a final concentration of 0.1% and let stand for 30 to 60 min. Although DEPC is a potent inhibitor of ribonucleases, it also causes the carboxymethylation of adenine bases and can result in inactivation of RNA. 4 Thus, any remaining traces of DEPC must be removed by autoclaving (DEPC decomposes into ethanol and CO2). The extremely short half-life of DEPC in Tris buffers renders treatment of these solutions ineffective. ~

Often overlooked is the fact that RNA is also highly susceptible to chemical degradation. It is important to avoid extremes in pH, high tem- peratures, and oxidizing agents. 6 At less than pH 5 some ribonucleases are highly active, whereas at high pH (> pH 9) RNA is subject to hydrolysis. Generally, RNA solutions should be maintained between pH 6.5 and 8.0, unless the RNA is being precipitated with ethanol (decrease to pH 5.2). It should be noted that at pH 7.6 poly(A) + RNA preferentially fractionates to the organic phase during phenol extractions. 7 This effect can be avoided by always extracting RNA with a 1 • 1 mixture of both phenol and chloro- form. 8 Also to be avoided when handling RNA are polyvalent cations (such as manganese and copper) which can cause hydrolysis of RNA. If pure water is not available, RNA should be stored in a solution of 1 - 2 m M EDTA. The rates of both chemical and enzymatic degradation are in-

4 L. Ehrenberg, I. Fedorcak, and F. Solymosy, Prog. Nucleic Acid Res, MoL Biol. 16, 185 (1974).

s S. L. Berger, Anal Biochem. 67, 428 (1975). 6 R. M. Buck, this series, Vol. 12A, p. 218. 7 G. Brawerman, J. Mendecki, and S. Y. Lee, Biochemistry 11,637 (1972). 8 R. P. Perry, J. La Torte, D. E. Kelley, and J. R. Greenberg, Biochim. Biophys. Acta 262, 220

(1972).

300 ExPRESSmN oF IOr~ CHANNELS [ 17]

creased at elevated temperatures. Thus, thawed RNA samples should always be kept on ice, and stocks should be stored frozen at - 80°.

Phenol must be equilibrated with aqueous buffer prior to use. Thaw a 100-g bottle of redistilled phenol [Bethesda Research Laboratories (BRL), Gaithersburg, MD, 5509UA] in a water bath at 65 °. Add a baked stir bar and approximately 100 ml of ribonuclease-free water. Add 20 ml of 2 M Tris (pH 7.5) and mix at room temperature for 20 min. Let the phases separate and then check the pH of the upper aqueous layer. If necessary adjust the pH to 7.5 with 5 M sodium hydroxide. The saturated phenol is stable at 4 ° for 3 to 4 weeks. For longer periods it should be stored in aliquots at - 2 0 °. If the phenol ever becomes pink or yellow it should be discarded. It is sometimes recommended that 8-hydroxyquinoline be added to a concentration of 0.1% to block oxidation of the phenol. In fact, this rather noxious agent only slows the oxidation process and, given that it is yellow in color, makes it impossible to determine when the phenol has become oxidized. Formamide should be deionized with a mixed bed resin (AG 501-X8, BioRad, Richmond, CA). Briefly, using a baked stir bar and beaker, mix 5 g of resin into 100 ml of formamide. Let stir for 30 to 60 min and then filter through Whatman (Clifton, N J) No. 1 paper. Store the deionized formamide in aliquots at -200.

Isolation of Biologically Active RNA

All cellular RNA purification protocols basically consist of two major steps: (1) initial homogenization in a solution which is designed both to disrupt cellular constituents, including RNA-protein complexes, and to inactivate ribonucleascs rapidly, and (2) separation of cellular RNA from DNA, proteins, and polysaccharides. The homogenization solutions used in various procedures include strong chaotropic agents such as guanidi- nium thiocyanate and guanidine hydrochloride, moderately denaturing agents such as phenol and urea, and detergents such as sodium dodecyl sulfate (SDS) and sarkosyl. Historically, the denaturant was chosen to reflect the Icvel of ribonucleasc endogenous to the source tissue. In prac- tice, however, it is prudent to use strong ribonuclease inhibitors regardless of the level of ribonuclcase expected. Total cellular RNA can be separ- ated from other cellular constituents by selective precipitation of RNA or by density gradient centrifugation. Although both separation techniques are effective, it should be noted that methods which rely on selective precipitation can result in varying yields, depending on such factors as thc nature of the starting material and the degree of initial homogenization and subsequent sheafing of cellular DNA.

In our expericnce, although may procedures yield RNA which is suit-

[17] TISSUE R N A AS SOURCE OF ION CHANNELS 301

able for in vitro manipulations, they do not always result in RNA which is translatable in Xenopus oocytes. The following two protocols give good yields of high molecular weight RNA that is translatable in oocytes, 9'1° It is best to use fresh tissue; however, satisfactory results have also been ob- tained using samples quick frozen in liquid nitrogen and then stored at - 80 ° until required.

Guanidinium Thiocyanate/Cesium Chloride Method

The following method is modified from the protocol of Chirgwin el al.ll and works well for RNA isolation from a variety of tissues and cell lines. The method is also useful for processing large numbers of samples.

1. Prepare 100 ml of GTC solution [4M guanidinium thiocyanate, 25 m M sodium citrate (pH 7.0), 0.5% N-laurylsarcosine]. Add 0.7 ml of 2-mercaptoethanol just prior to use (final concentration is 100 mM).

2. Prepare a solution of 5.7 M CsCI containing 25 mM sodium acetate (pH 6.0) and 5 m M EDTA. Sterilize by filtration.

3. Homogenize the tissue or cells at room temperature in 10- 1 5 ml of GTC solution per gram of sample. A Dounce homogenizer can be used for soft tissues and for cell lines, whereas a Polytron is often required for homogenization of fibrous tissues such as skeletal muscle. After homogen- ization, transfer the sample to a sterile centrifuge tube and remove any particulate material by centrifugation at 4000 rpm for 10 min at room temperature.

4. Pipette 3.5 ml of CsC1 solution into a 14 × 89 mm polyallomer ultracentrifuge tube. Carefully layer the GTC supernatant onto the CsC1 cushion and balance opposing tubes.

5. Centrifuge the SW41 rotor at 32,000 rpm for 20 hr at 20 °. 6. Carefully aspirate off the GTC and CsC1 until just near the bottom

of the tube. Decant offthe remainder of the solution, and, keeping the tube inverted, cut off the tube below the CsC1 cushion interface.

7. Resuspend the pellet in 250 gl of TE-SDS (10 mM Tris, pH 7.5, 1 m M EDTA, 0.1% SDS) and transfer to a sterile 1.5-ml microcentrifuge tube. Rinse the untracentrifuge tube with 150/tl TE- SDS and pool.

8. Precipitate the RNA by addition of 50/~1 of 3 M sodium acetate (pH

9 j, S. Trimmer, S. S. Cooperman, S. A. Tomika, J. Zhou, S. M. Crean, M. B. Boyle, R. G. Kallen, Z. Sheng, R.L. Barchi, F. J. Sigworth, R. H. Goodman, W. S. Agnew, and G. Mandel, Neuron 3, 33 (1989).

1o T. P. Snutch, J. P. Leonard, M. M. Gilbert, H. A. Lester, and N. Davidson, Proc. Natl. Acad. Sci U.S.A. 87, 3391 (1990).

~ J. M. Chirgwin, A. E. Przygyla, R. J. MacDonald, and W. J. Rutter, Biochemistry 18, 5294 (1979).

302 ~XPR~SSION OF ION CHArnELS [ 17]

5.2) and 2.5 to 3 volumes of ethanol. Incubate at - 2 0 ° overnight or on dry ice for 15 min.

9. Pellet the RNA in a microfuge for 15 min at 4 °. Remove the ethanol, wash once with 80% ethanol, and dry the pellet only enough to remove the last traces of ethanol. Dissolve the pellet in ribonuclease-free water at a concentration of I to 2 mg/ml. Store in aliquots at - 80 °.

Lithium Chloride~Urea Method

The following method is modified from the procedure of Auffray and Rougeon n and has the advantages that it does not involve a lengthy centrifugation through CsC1 and that it can conveniently be used for large-scale RNA preparations (up to 50-60 g tissue). Total RNA isolated using this method may be contaminated by small amounts of genomic DNA (l -2%). If the RNA is to be utilized for polymerase chain reaction (PCR) analysis and primers which could distinguish between RNA and DNA products are not available, then the RNA should first be treated with ribonuclease-free DNase.

1. Prepare a solution of 6 M urea, 3 M lithium chloride, 0.5% N-lau- rylsarcosine, and 10 m M sodium acetate (pH 5.2). Cool to 4 ° prior to use.

2. Homogenize the tissue or cells with a precooled Dounce homogen- izer in a volume of 8 to 10 ml per gram of tissue or per milliliter of packed cells. The solution will become quite viscous, and it is important to shear the DNA in order to maximize the amount of RNA recovered. Approxi- mately 35 to 45 strokes is sufficient.

3. Place the mixture on ice in the cold overnight. Pellet the RNA in a high-speed centrifuge using baked Corex tubes at 8000 rpm for 30 min at 4 °"

4. Carefully decant off the supernatant and, with the tube inverted, wipe the sides of the tube with a tissue. Dissolve the pellet in 1- 2 ml of cold 10 m M Tris (pH 7.5), 1 m M EDTA, 0.5% sarkosyl and transfer to a sterile disposable polypropylene tube. Pipetting the sample up and down several times is usually sufficient to dissolve the pellet.

5. Extract the sample with 1 volume of buffer-saturated pheno-Sevag (1:1; Sevag is chloroform-isoamyl alcohol, 24:1). For large-scale RNA preparations allow the sample to mix on a shaker in the cold for 15 min. For small-scale RNA preparations it is sufficient to add cold phenol-Sevag and mix by hand for 1 - 2 min. Separate out the phases by centrifugation in a 4 ° centrifuge for 5 min at 5000 rpm. Small-scale preparations can be accommodated in a microfuge.

12 C. Auffray and F. Rougeon, Eur. J. Biochem. 107, 303 (1980).

[17] TISSUE RNA AS SOURCE OF ION CHANNELS 303

6. Transfer the upper aqueous layer to a clean tube and extract twice more with phenol-Sevag.

7. Extract the aqueous sample with 1 volume of Sevag and centrifuge as in step 5.

8. To the aqueous layer add l / l0 volume of 2.5 M sodium acetate (pH 5.2) and 2.5 volumes of ethanol. Store at - 2 0 ° overnight and then recover the RNA by centrifugation at 8000 rpm for l0 min.

9. Wash the RNA pellet once with 80% ethanol and centrifuge as in Step 8. Discard the ethanol, dry the pellet briefly, and dissolve the RNA at a concentration of 1 to 2 mg/ml in ribonuclease-free water. At this point the RNA is pure enough to perform Northern blots and to isolate poly(A) + RNA using atfinity chromatography. For in vitro translation, PCR analy- sis, or injection into Xenopus oocytes, the total RNA should be reprecipi- tated as in Steps 8 and 9.

Prior to using the total RNA for oocyte injections the quality of the RNA should be examined. The absorbance ratio A26o/A280 of the purified RNA should be between 1.9 and 2.1. Lower ratios are usually indicative of either protein contaminants or incompletely removed reagents (e.g., sarko- syl and phenol both absorb light in the 260-280 nm range). The RNA should also be checked visually on an 0.8 to 1.0% nondenaturing agarose gel containing 1 #g/ml ethidium bromide. Alternatively, if an RNase-free gel box is not available, the RNA can be separated through a 0.8 to 1.1% agarose gel containing 1.1 M formaldehyde (see below). The total RNA should show two prominent bands representing the 28 and 18 S ribosomal RNAs. The relative intensity of the 28 to 18 S bands should be approxi- mately 2: l, and there should be minimal smearing of lower molecular weight RNAs.

Isolation of Poly(A) + RNA

Most eukaryotic mRNAs contain stretches of 50 to 200 adenylic acid residues at the 3' end [poly(A) ÷ mRNA]. Commercially available cellulose resins which have covalently attached poly(dT) residues [oligo(dT)-cellu- lose type 3, Collaborative Research, Bedford, MA] can be used to isolate selectively mRNA from total cellular RNA. 13 With a small number of samples poly(A) + RNA can be isolated using a column chromatography protocol [columns either can be homemade, consisting of a sterile syringe plugged with baked glass wool, or they can be purchased already packed

13 j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual," 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989.

304 EXPRESSION OF ION CHAr~EBS [ 17]

with oligo(dT)-cellulose; Sigma, St. Louis, MO]. For multiple RNA sam- ples or when starting from small amounts of total RNA (<2 mg) it is practical to use a batch isolation protocol23

Poly(A) + RNA can also be purified using poly(U)-Sepharose (Pharma- cia, Piscataway, N J). Owing to the collapsible nature of the matrix, only column chromatography can be performed with poly(U)-Sepharose. 14 Poly(U)-Sepharose columns have a much quicker flow rate than oligo(dT) columns and, additionally, the matrix binds poly(A) + RNA more effi- ciently than oligo(dT)-cellulose.

1. Equilibrate 1 g of poly(U)-Sepharose in 10 ml of 1 × binding buffer (1 × binding buffer is 0.4 M NaC1, 10 m M Tris, pH 7.5, 1 m M EDTA, 0.1% SDS).

2, Prepare a column using a 10-ml disposable pipette in which the bottom has been plugged with baked glass wool. Form a column with the equilibrated poly(U)-Sepharose and then wash through a further 15 ml of 1 × binding buffer.

3. Heat the RNA sample (in sterile water or 1 m M EDTA at a concen- tration of 0.5 to 1 mg/ml) to 80 ° for 1 rain. Cool on ice and then add 1 volume of 2 X binding buffer.

4. Pass the RNA mixture over the column and collect the elutant in a ribonuclease-free tube. Pass the elutant over the column once more and then wash the column with 10 ml of I X binding buffer.

5. Elute nonspecifically bound RNA by passing 20 ml of low salt buffer over the column (low salt buffer is 10 m M Tris, pH 7.5, 1 m M EDTA, 0.1% SDS).

6. Elute the poly(A) + RNA by passing 4 ml of room temperature 80% formamide (dionized), 10 m M Tris, pH 7.5, 1 m M EDTA through the column and collect the elutant in a sterile tube. Dilute the formamide by addition of 2 volumes of sterile water and precipitate the RNA by addition of 1/10 volume of 2.5 M sodium acetate (pH 5.2) and 2.5 volumes of cold ethanol. Incubate at - 2 0 ° overnight and then recover the poly(A) + RNA by centrifugation at 10,000 rpm for 20 min. Wash the RNA once with 80% ethanol, dry briefly, and then dissolve in ribonuclease-free water at a concentration of 1 mg/ml. Typically, 1.5-2% of the total RNA will be recovered as poly(A) +.

The microinjection of exogenous RNA into Xenopus oocytes is de- scribed in [14] (in this volume). Under most circumstances robust re- sponses are obtained by injection of approximately 100 to 200 ng of total RNA per oocyte (50 nl of a 2-4/ tg/ / t l RNA solution). Injection of 50

t4 A. Jaeobson, this series, Vol. 152, p. 254.

[ 17] TISSUE RNA AS SOURCE OF ION CHANNELS 305

ng of poly(A) ÷ RNA is sufficient for detection of many ion channels and neurotransmitter receptors. For reasons that are at present unclear, the injection of poly(A) + RNA results in responses that are only 3 to 4 fold greater than that obtained from injection of a similar amount of total RNA [a 10 to 12-fold increase in signal would be expected by poly(A) + enrich- ment]. However, in many instances the increase in signal obtained with poly(A) + RNA justifies the additional effort required for purification.

Denatur ing Gel Electrophoresis and Nor thern Blot Analysis

Prior to injection into oocytes the quality of the purified RNA should be examined. First, the RNA should be examined visually for degradation using formaldehyde gel electrophoresis (Steps 1 to 5 below). Second, the RNA should be transferred to a nylon membrane and then hybridized either to a probe encoding the molecule under study (if available) or to a probe which is known to be expressed in the source tissue and is encoded by a relatively large RNA transcript (Northern bloPS). For example, hy- bridization of a rat brain sodium channel cDNA probe to intact brain RNA should result in strong hybridization to 9- to 10-kb transcripts and show little evidence of a low molecular weight smear. If a probe is available for the gene under study, Northern blot analysis can also provide impor- tant information with respect to the size of the mRNA(s) encoding the gene product, the number of related mRNAs (usually an indication of gene families or alternative splicing), and the relative abundance of the tran- script. In addition, Northern blot analysis using RNAs isolated from various tissues and developmental stages can give an indication as to which source will provide the most robust signal on injection of RNA into oocytes.

The following is a recipe for 150 ml of a 1% agarose gel containing 1.1 M formaldehyde.

1. Prepare 500 ml of 10X MOPS buffer (10× MOPS is 0.2 M MOPS, pH 7.0, 80 m M sodium acetate, 10 m M EDTA). Filter sterilize and store in a dark bottle at 4 °.

2. Add 1.5 g of ultrapure agarose to 122 ml of distilled water and dissolve by heating in a microwave. Add 15 ml of 10× MOPS buffer and cool to 60 ° in a water bath. After adding a baked stir bar to the flask, pipette in 13.4 ml of formaldehyde and stir for 15- 20 sec. Pour the gel in a fumehood and let set for 1 hr to overnight.

3. While the gel is setting prepare the RNA samples for loading. For

15 p. Thomas, Proc. Natl. Acad. Sci. U.S.A. 77, 5201 (1980).

306 EXPRESSION OF ION CHANNELS [ 1 7]

each RNA sample mix 5 pl of 10× MOPS, 8.75 gl of formaldehyde, 25/tl of deionized formamide, and the RNA sample (in a volume of 11.25 gl). Use 25 - 30/tg of total RNA or 1 - 5 gg ofpoly(A) + RNA per lane. Heat the sample to 65 ° for 15 min and place on ice for 2 min. Add 2/ t l of RNA loading buffer (50% glycerol, 10% Ficoll, 1 m M EDTA, 0.4% bromphenol blue, 0.4% xylene cyanol). For markers use 5/tg of synthetic RNA ladder (0.24 to 9.5 kb; BRL 5620SA). Treat the markers as above except that prior to heating add 1 #1 of 1 mg/ml ethidium bromide. This obviates the need to stain the gel after running.

4. RNA bands will be fuzzy if, prior to loading the gel, the wells are not rinsed with 1 × MOPS running buffer. Load the samples and electrophor- ese at 75- 80 V until the bromphenol blue just reaches the bottom of the gel (4-5 hr for a 15-cm gel).

5. Carefully remove the gel and take a picture of the RNA markers with a ruler laid alongside as a reference.

6. Soak the gel in 1 × TAE (40 mMTris-aceta te , 1 mMEDTA) for 15 min to remove most of the formaldehyde. Transfer the gel to a nylon membrane [Durlon, Strategene (La Jolla, CA); Nytran, Schleicher & Schuell (Keene, NH); Hybond-N, Amersham (Arlington Heights, IL)] by either capillary blot overnight or by electroblot using a commercial appa- ratus (Hoefer, San Francisco, CA). Fix the RNA to the membrane by UV cross-linking as described by the supplier.

7. Hybridization and washing conditions will depend on the character- istics of the probe and target sequences} 6 Generally, DNA probes are labeled by nick-translation or random priming, and hybridization is carried out at 42 ° in 50% formamide and an aqueous mixture consisting of 5X SSPE (1 × SSPE is 0.18 M NaC1, 10 m M sodium phosphate, pH 7.4, 1 m M EDTA), 0.5% SDS, 0.2 mg/ml sheared, denatured salmon sperm DNA, 0.1 mg/ml yeast tRNA, and 2 .5× Denhardt's [1X Denhardt's is 0.02% bovine serum albumin (BSA), 0.02% Ficoll, 0.02% polyvinylpyrroli- done]. Alternatively, hybridizations can be carried out at 68 ° to 70 ° in aqueous hybridization buffer alone. For synthetic RNA probes the high stability of R N A - R N A hybrids requires that hybridization be done under more stringent conditions, for example, in 50% formamide at 68 °. Blots should be washed at 10 ° to 12 ° below the calculated T m of the probe. At lower stringency hybridization and wash conditions nonspecific bands can appear, making the results unclear or misleading. In general, a low signal-to-noise ratio is obtained using short (<50 bases) oligonucleotide probes.

,6 G. H. Keller and M. M. Manak, "DNA Probes." Stockton Press, New York, 1989.

[17] TISSUE RNA AS SOURCE OF ION CHANNELS 307

Ribonuclease Protection

In addition to Northern blot hybridization, another method of analyz- ing low abundance mRNAs, characteristic of most mammalian ion chan- nels and receptor mRNAs, is by RNase protection? 7 This method is an extremely sensitive measure of mismatch produced between two distinct mRNAs and is therefore quite useful for discriminating between mRNAs produced by members of a multigene family. It is also useful for detection of the transcription start sites of mRNAs or for determination of intron/ exon boundaries. The assay is based on the differential sensitivities of single-stranded and double-stranded nucleic acids to cleavage by certain ribonucleases. Specifically, "exactly matched" RNA hybrids are protected from digestion with ribonucleases A and T 1, whereas regions of mismatch in imperfect hybrids are digested by these same ribonuclease activities. When the sequence of the RNase protection probe is known precisely, the size of the resulting "protected" fragments clearly reveals, by fractionation on denaturing sequencing gels, whether the probe and test mRNAs are identical or not. The primary advantage of the RNase protection technique over Northern blot analysis is that the former is much more sensitive. On the other hand, only Northern blot analysis can reveal the size of the mature test mRNA.

1. Antisense RNA probes are synthesized in vitro using either a com- mercial kit (Riboprobe, Promega, Madison, WI) or using the following protocol. Mix the following at room temperature: 2 #l of l0 X transcription buffer, 2/zl of 5 mM ATP, 2/zl of 5 mMGTP, 2#1 of 5 mM CTP, 0.5#1 of 0.5 M dithiothreitol, 0.5/zl RNasin (30-40 U//zl), l0/~Ci [a-32p]UTP (400-800 Ci/mmol), l #l linearized plasmid DNA (l #g//zl), and 1 #l of SP6, T3, or T7 RNA polymerase (30-40 U/#l). Incubate the sample at 37 ° for 1 hr. The 10× transcription buffer is 200 mM Tris (pH 7.5), 60 mM MgC12, 20 mM" spermidine-HC1, and 100 ~ NaC1. The cold nucleotide stocks are made up in sterile 70 mM Tris (pH 7.5).

2. Add 2 #l ofDNase (RNase-free; 1000 U/ml or 1 mg/ml) and incu- bate at 37 ° for 15 min.

3. Bring the volume of the reaction to 100 #l with sterile water and extract once with 40 #l of phenol- Sevag ( l : 1), Vortex briefly and separate the phases by spinning in a microfuge for 3 min.

4. Remove the unincorporated radiolabeled nucleotides by spin col- umn chromatography and precipitate the probe by addition of 10 #g carrier tRNA (Sigma), 0.1 volumes of 2.5 M sodium acetate, and 2.5 volumes of ethanol.

~7 K. Zinn, D. Dimaio, and T. Maniatis, Cell (Cambridge, Mass.) 34, 865 (1983).

308 EXPRESSION OF ION CHANNELS [ 17]

5. Recover the probe by centrifugation and dissolve in 10/~1 of RNA gel buffer. Heat the sample to 90 ° for 5 min and run on a 6% polyacrylam- ide sequencing gel at 40-50 mA until the bromphenol blue is near the bottom of the gel.

6. Place the gel on a glass plate and cover with Saran wrap. Place a second glass plate on top of the wrapped gel and carry the sandwich to the darkroom. Remove the top glass plate and turn the gel face down onto a piece of film. Outline the glass plate with a marker and develop the autoradiogram. An exposure of 15-25 sec is sufficient to see the RNA band. Using the autoradiograph as a guide, excise the correct band and transfer to a sterile microfuge tube. Freeze the gel piece and then mince with a baked spatula. Add 1 ml of elution buffer (0.5 M ammonium acetate, 1 mMEDTA, 0.1% SDS) and shake at 37 ° for 4 - 6 hr.

7. Recover the eluted RNA by spinning the gel mixture in a micro- fuge at 4 ° for 15 min. Transfer the supernatant to a fresh tube and re- peat the centrifugation. To the supernatant, add l0/zg of carder tRNA and precipitate the probe with 2.5 volumes of ethanol. An alternative protocol for recovering the RNA is as follows: after the incubation in gel elution buffer pass the gel mixture through a prewetted Elutip-r prefilter (Schleicher & Schuell) and subsequently recover the RNA by ethanol precipitation.

8. Resuspend the probe in 25 to 50/tl of 5 X hybridization buffer (200 m M PIPES, pH 6.4, 2 M NaC1, 5 m M EDTA) and count an aliquot in a scintillation counter. Adjust the volume of the probe so that there is 5 X 105 counts/min (cpm) per 6/zl of hybridization buffer. Add 24/zl of deionized formamide for each 6/zl of probe to yield the final hybridization mixture.

9. For each RNA sample to be analyzed, precipitate I0-50/zg of total or l - l0/zg of poly(A) + RNA and resuspend in l0/zl of hybridization mixture. Heat the samples at 90 o for 5 min and then transfer to the final hybridization temperature (between 45 o and 55 o) for 8 - 16 hr.

10. To each sample add 350/zl of RNase solution (40/zg/ml RNase A, 2/zg/ml RNase T1 in l0 m M Tris, pH 7.5, 300 m M NaC1, 5 m M EDTA) and incubate at 30 ° for l - 2 hr. Destroy the RNase by addition of 20/zl of 10% SDS and 2.5/zl of proteinase K (20 mg/ml in water), then incubate at 37 ° for 15-60 rain.

11. Extract the samples with 350/tl of phenol-Sevag and precipitate by addition of 10/tg of cartier tRNA and 1 ml of ethanol. Resuspend the pellet in 10/zl of gel loading buffer (80% formamide, 1 m M EDTA, 0.1% bromphenol blue, 0.1% xylene cyanol) and heat to 90 ° for 5 min prior to analyzing on a 6% acrylamide sequencing gel. Typical autoradiograph exposure times are 12 to 48 hr.

[I 7] TISSUE R N A AS SOURCE OF ION CHANNELS 309

To demonstrate that protection has occurred, a sample of untreated probe should be run alongside the RNase-treated samples. Radiolabeled DNA size markers should also be included on the gel. A convenient way both to determine the sensitivity of the protection reaction and to quanti- rate the amount of target sequence in an RNA population is to generate nonlabeled sense RNA in vitro from the transcription plasmid containing the probe and then to titrate defined amounts of sense RNA against the radiolabeled antisense probe.

Other Considerations

Although there are numerous instances of the successful expression of ion channels and neurotransmitter receptors in Xenopus oocytes, it is still difficult if not impossible to detect the functional expression of certain conductances. The list of cloned receptors and channels which are poorly expressed in oocytes includes the eel sodium channel, ~8 the rabbit skeletal muscle L-type calcium channel ~9 and the human brain serotonin 1A re- ceptor. 2° There are a number of possible explanations for inefficient or improper translation in oocytes. It is possible that the mRNA encoding the molecule under study is quite rare. In such instances, enrichment tech- niques such as poly(A) + RNA isolation and RNA fractionation methods (see [ 16] in this volume) may alleviate the problem. It may also be that the 5' noncoding region of the mRNA under study is actually detrimental to translation in ovo. Indeed, the enzymatic removal of upstream sequences has been shown to improve expression in oocytes dramatically. 2~ In a number of other instances, the conductances induced in oocytes by exoge- nous RNA show alterations from that found in the wild-type tissue. For example, whereas the Shaker A channel is insensitive to charybdotoxin in insect cells, it is blocked by nanomolar concentrations of this toxin when expressed in oocytes. 22 Another example is the finding that the Xenopus oocytes do not perform the proper N-linked glycosylations of Torpedo nicotinic acetylcholine receptor subunits, 23 alluding to the fact that Xeno- pus oocytes do not always perform the correct posttranslational modifica- tions TM (see [45] in this volume).

18 W. B. Thomhill and S. R. Levinson, Biochemistry 26, 4381 (1987). 19 E. Perez-Reyes, H. S. Kim, A. E. Lacerda, W. Home, X. Wei, D. Rampe, K. P. Campbell,

A. M. Brown, and L. Birnbaumer, Nature (London) 340, 233 (1989). 2o A. Fargin, J. R. Raymond, M. J. Lohse, B. K. Kobilka, M. C. Caron, and R. J. Lefkowitz,

Nature (London) 33S, 358 (1988). 21 M. M. White, L. Chen, R. Kleinfeld, R. G. Kallen, and R. L. Barchi, Mol. Pharmacol. 39,

604 (1991). 22 R. MacKinnon, P. J. Reinhart, and M. M. White, Neuron 1, 997 0988). 23 A. L. Bullet and M. M. White, J. Membr. Biol. 115, 179 (1990).