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Agarose gel electrophoresis of denatured RNA with silver staining
ANALYTICAL
BIOCHEMISTRY
169, 132- 137 (I 988)
Agarose Gel Electrophoresis
of Denatured
RNA with Silver Staining
ROSE N. SKOPP AND LESLIE C. LANE Department
of Plant Patholqqy.
Llniversity
qf’Nehrasku.
Lincoln.
Nebraska
68583-07-72
Received June 18. 1987 This paper describes agarose gel electrophoresis and silver staining of denatured RNAs. Glyoxal- or formaldehyde-denatured RNAs are electrophoresed in an agarose gel cast on a plastic support using an inert low conductivity buffer. Following electrophoresis, the gel is stained with a sensitive silver stain. The method produces sharp, well-resolved bands and yields accurate RNA size estimates. Because of its sensitivity and simplicity, it is suitable for routine laboratory use. 8 1988 Academic Press. inc. KEY WORDS: gel electrophoresis; silver stain; RNA: denaturation; molecular weights,
Existing methods for separating denatured RNAs are inconvenient and insufficiently sensitive for routine laboratory use. A review of the literature illustrates the disadvantages of existing methods and suggests improvements. Gel electrophoresis is convenient for separating RNAs (1,2) and estimating their sizes (3). RNA migration rates depend on both molecular weight and conformation (4). Conformational heterogeneity will reduce RNA resolution and conformational differences among RNAs can limit the accuracy of molecular weight estimates. However, denaturants can minimize conformational differences, yielding sharper bands and allowing more accurate size estimates. The denaturants formamide (5) formaldehyde (6) and urea (7.8) reduce heterogeneity but do not completely eliminate conformational differences, whereas glyoxal (9) and methylmercury hydroxide eliminate both conformational differences and heterogeneity. However. these more effective denaturants have disadvantages. Glyoxal-denatured RNAs stain poorly with standard nucleic acid dyes and methylmercury hydroxide is both expensive and toxic. RNA can be electrophoresed in either polyacrylamide or agarose gels. Polyacryl0003-‘697/88
$3.00
CopyrIght t 1988 by Academic Press. Inc. All rights of rcprc&ct,on I” any form reserved
amide gels are relatively unsatisfactory because their pores are too small for large, denatured RNAs. Polyacrylamide is also chemically incompatible with some methods. For example. formaldehyde reacts with polyacrylamide and methylmercury hydroxide inhibits acrylamide polymerization. Agarose is an alternative gel matrix. It can easily be cast on a support medium and it contains large pores. Grades of agarose with high gel strength and low endosmosis are especially suitable for electrophoresis of RNA. Commercial agarose contains ribonuclease which must be inactivated before electrophoresis. Iodoacetate effectively inactivates the ribonuclease in commercial agarose (9). Standard methods for staining denatured RNA are ineffective. Ethidium bromide, the most commonly used stain, detects doublestranded nucleic acids with high sensitivity: however, it stains denatured RNA only faintly. Acridine orange stains denatured RNA (lo), but gives a high background. Silver stains detect double-stranded nucleic acids with high sensitivity (1 1) and in our experience are equally satisfactory for denatured RNA. This paper presents a method for reliable separation and size estimation of RNA dena-
132
RNA ELECTROPHORESIS
tured with formaldehyde or glyoxal. Nucleic acid is separated in agarose cast on GelBond film using an inert, low conductivity electrophoresis buffer containing iodoacetate to inactivate ribonuclease. The silver stain of Willoughby and Lambert (12) detects denatured RNA with high sensitivity. The procedure is simple. rapid, and sensitive. MATERIALS
AND METHODS
Materials GelBond film and low endosmosis (LE)’ high gelling temperature (HGT) agarose were obtained from the FMC Corp. (Rockland. ME). The horizontal gel apparatus was built from lucite with a gel stage of 90 X 110 mm. Reagent grade formaldehyde (Fisher brand, 37%, w/w) containing methanol stabilizer and glyoxal trimeric dihydrate (Sigma, approximately 98%) were used without additional purification. Tungstosilicic acid was purchased from Gallard-Schlesinger Chemical Mfg. Corp. (Carle Place, NY). Other chemicals were reagent grade where available. Methods Viruses were purified by standard procedures (13). Purified preparations were diluted to approximately 0.5 mg/ml with 0.1 M Tris containing 0.05 M HCl. The solution was brought to 3% (w/w) in sodium dodecyl sulfate (SDS). One volume of water saturated phenol was mixed with 1 vol of virus (400-J aliquots) and heated for 5 min at 60°C. The sample was cooled and centrifuged for 5 min at 8OOOg. The upper (aqueous) phase was withdrawn and precipitated with 2-3 vol of reagent grade ethanol for 2 h at -20°C or for 1 h at -80°C. Recovery of the aqueous phase ’ Abbreviations used: LE, low endosmosis; HGT, high gelling temperature: SDS, sodium dodecyl sulfate; DMSO, dimethyl sulfoxide; TMV, tobacco mosaic virus; BMV, brome mosaic virus: BSMV. barley stripe mosaic virus: GC, guanine-cytosine.
133
was about 50% of the initial aqueous volume. The suspension was centrifuged for 10 min at 8OOOg. The supernatant was discarded and the pellet was dried under vacuum. Escherichia coli total nucleic acids were extracted by standard methods ( 14). Formaldeh~~de modijication. Vacuumdried RNA pellets were dissolved in about 100 ~1 of 2% (w/w) SDS, 10% (w/v) sucrose, 2 mM Na,-EDTA, 75 mM N-ethylmorpholine, and 50 mM phosphoric acid. Formaldehyde was added to a final concentration of 3.7% (w/w). The sample was heated for 10 min at 70°C. Small aliquots (3-10 ~1) were electrophoresed. C;lJv.t-al mod$cation. Vacuum-dried RNA pellets were dissolved in approximately 100 ~1 of a solution containing 1 M glyoxal. 75 mM iii-ethylmorpholine, 50 mM phosphoric acid, and 50% (v/v) dimethyl sulfoxide (DMSO). The sample was heated for 10 min at 70°C. Small aliquots (3-10 ~1) were electrophoresed. Both formaldehydeand glyoxal-modified samples may be stored for several months at -30°C . Ckl pwparation. HGT agarose was dissolved by heating in 1%’ (w/v) 75 mM h’-ethylmorpholine, 50 mM phosphoric acid. 0. l?ic (w/w) SDS, and 8 mM iodoacetic acid: 10 ml was poured onto 85 X loo-mm GelBond film. A 1.25-mm-thick polyethylene comb with 4-mm teeth was inserted into the gel and held in place with tape. After the agarose solidified (at least 10 min) the comb was carefully removed. Note that sample wells are difficult to locate once the gel is submerged. One method to facilitate loading is to mark well positions on the back of the plastic support. Prior to electrophoresis, the gel was submerged and incubated for 1 h in electrophoresis buffer (75 mM N-ethylmorpholine, 50 mM phosphoric acid. 1.85% (w/w) formaldehyde). Samples were applied and the gel was electrophoresed at a current flux of about 12 mA/cm’ for 90 min. Silver stain. Following electrophoresis, the gel was stained by the procedure of Wil-
134
SKOPP AND LANE
loughby and Lambert ( 12). The gel was fixed for 15 min in a solution containing 17.3 g of sulfosalicylic acid, 25 g of trichloroacetic acid, 25 g of zinc sulfate, and 500 ml of distilled water. After washing the gel three times for 10 min each in distilled water, it was dehydrated for 5 min in ethanol. The gel was dried with a heat lamp or hot air dryer. The silver stain was prepared from two stock solutions, A and B. Solution A contained 25 g of anhydrous Na2C03 and 500 ml of distilled water. Solution B consisted of 1 g of NH4N03, I g of AgN03, 5 g of tungstosilicic acid, 7.0 ml of 37% (w/w) formaldehyde, and 500 ml of distilled water. To stain, 13.5 ml of solution B was mixed with 6.5 ml of solution A. The dried gel was immersed in the staining bath and vigorously agitated. A white precipitate, which gradually darkened to gray, formed in the solution. When this precipitate became dark gray or when bands first appeared, the gel was removed. rinsed briefly (3-4 s) in distilled water, and placed in fresh staining solution. Generally, two staining cycles were sufficient but lightly loaded gels required three. Staining was stopped by placing the gel in 1WI(v/v) acetic acid for about 5 min. The gel was then rinsed briefly in distilled water and dried by evaporation. RESULTS
Both formaldehydeand glyoxal-denatured RNAs electrophoresed as distinct bands on agarose gels. Figure 1 shows RNAs from tobacco mosaic virus (TMV), brome mosaic virus (BMV), and barley stripe mosaic virus (BSMV) denatured with formaldehyde and glyoxal. Note that glyoxal-modified RNAs migrated slightly slower than formaldehyde-modified counterparts, suggesting that they have more extended configurations. Similarly, Fig. 2 shows the 16 and 23 S 6. coli rRNAs denatured with formaldehyde and glyoxal. Mixtures of BMV and TMV RNAs denatured with formaldehyde or glyoxal served as size standards.
a
b
c
d
FIG. I Formaldehyde- and glyoxal-denatured RNAs electrophoresed on a horizontal 1% agarose gel stained with silver. (a) TMV and BMV RNAs denatured with formaldehyde. (b) TMV (6397 nucleotides) and BMV RNAs (3234, 7865. 21 14. and 876 nucleotides) denatured with glyoxal. (c) BSMV RNAs (3890. 3289, and 279 I nucleotides) denatured with formaldehyde. (d) BSMV RNAs denatured with glyoxal. Heavily stained bands contain approximately 50 ng of RNA.
Both formaldehyde and glyoxal methods gave accurate molecular size estimates. A graph of the logarithm of the molecular size versus mobility gave a straight line. The slope of this line depended on the electrophoretic conditions and differed between formaldehyde and glyoxal-denatured RNAs. Standard curves were constructed using previously established values for the sizes of TMV (6397 nucleotides (15)) and BMV RNAs (3234. 2865. 2114. and 876 nucleotides ( 16)). The molecular sizes of BSMV RNAs 2 and 3, and E. coli 16 and 23 S rRNAs estimated from these curves using regression analysis. were consistent with nucleotide sequences ( 17-20). The correlation coefficients ranged from 0.992 to 0.999. To obtain reliable size estimates, standards and samples must be treated with the same denaturant. Table 1 compares published estimates for RNA molecular sizes with those determined by the glyoxal and formaldehyde methods described here.
RNA a
b
c
135
ELECTROPHORESIS
d
FIG. 2. Formaldehydeand glyoxal-denatured RNAs electrophoresed on a horizontal 1% agarose gel stained with silver. (a) E. co/i rRNAs (2904 and IS41 nucleotides) denatured with formaldehyde (the band closest to the origin is DNA). (b) TMV (6397 nucleotides) and BMV RNAs (3234, 2865, 21 14. and 876 nucleotides) denatured with formaldehyde. (c) E. co/i rRNAs denatured with glyoxal. (d) TMV and BMV RNAs denatured with glyoxal. Heavily stained bands contain approximately 50 ng of RNA.
In early experiments, RNA was denatured with formaldehyde in the presence of Ficoll. However, this produced indistinct, poorly defined RNA bands which smeared toward the origin (Fig. 3). Adding Ficoll after formaldehyde denaturation produced similar patterns. Extended incubation of RNA with formaldehyde and Ficoll progressively increased smearing and retarded migration (data not shown). Replacing Ficoll with 10%) (w/v) sucrose eliminated this problem. Ficoll did not interfere with electrophoresis of glyoxal denatured RNAs.
(21)) suggested that formaldehyde does not completely denature GC-rich RNAs. Since none of the RNAs discussed here were GC rich, it is unwise to assume that formaldehyde will completely denature all RNAs. Nonetheless, this procedure yields accurate molecular weights for BSMV RNAs where earlier formaldehyde methods gave high values (22). Two explanations can be entertained for the inaccuracy of earlier values: reversibility of formaldehyde modification and interaction of formaldehyde-modified RNA with polyacrylamide. Omitting formaldehyde from the running buffer does not influence the apparent size of BSMV RNAs in our system (data not shown). This suggests that polyacrylamide may be an unreliable medium for electrophoresis of formaldehyde-modified RNAs. The apparent crosslinking of RNA to Fico11 by formaldehyde also suggests potentially undesirable aldehyde chemistry. If formaldehyde crosslinks RNA to Ficoll, it likely crosslinks RNA to sucrose. The sucrose adduct would be small and unlikely to influence electrophoretic properties of the RNA.
TABLE COMPARISON
OF PUBLISHED
MOLECULAR WEIGHTS THE GLYOXAL AND
I ESTIMATES
WITH THOSE FORMALDEHYDE
FOR RNA
DETERMINED METHODS
~1’
Sire in nucleotides RNA BSMV- I BSMV-2 BSMV-3 E coli(l6S) E. cdi (23 S)
Glyoxal 3910 3250 2700 l52Ok 3910
+ 210 i I40 + 140 60 f 80
Formaldehyde 3830 3270 2720 15502 2850
r 120 If I30 t 150 90 f 130
Published 3890” 3289(17) 2791 (IX) 1541 (19) 2904 (20)
DISCUSSION
This procedure combines several techniques to give simple, high resolution separation of denatured RNA on horizontal agarose gels. Both glyoxal and formaldehyde derivatives yield accurate molecular weight estimates. Earlier studies (for example, Ref.
Note. RNA sizes were determined from standard curves derived using denatured RNAs of known size. e.g., TMV and BMV. Standards and samples were treated with the same denaturant. Each value represents the average of at least five separate determinations using different gels. ’ Estimate based on the chemical sequence of RNA-3 (17) and particle length ofvirions (23).
136
SKOPP
AND
Nonetheless, crosslinking of nucleic acids to carbohydrates could influence other applications of formaldehyde, Reagents have been chosen for convenience. Both the anion and cation of the buffer have low ionic mobility and are inert to aldehydes. Any buffer which satisfies these criteria should be satisfactory. Both A’-ethylmorpholine and phosphoric acid are liquids and can be dispensed volumetrically. The buffer neutralizes acids which could interfere with RNA modification. If buffering is insufficient, acids can be neutralized by adding more N-ethylmorpholine. In our experience. ion-exchange purification of glyoxal is unnecessary. The thickness and composition of the well-forming comb influence the sharpness of RNA bands. Teflon lucite, and polyethylene combs varying in thickness from 0.75 to 2.5 mm were tried. In general, thinner combs gave sharper bands but wells formed by 0.75-mm combs were difficult to load. a
b
c
FIG. 3. Formaldehyde-denatured RNAs from TMV (6397 nucleotides) and BMV (3234, 2865, 21 14. and 876 nucleotides) electrophoresed on a horizontal 1% agarose gel and stained with silver. (a) 10% (w/v) Ficoll added to the sample after denaturation. (b) 10% (w/v) Ficoll present during sample denaturation. (c) 10% (w/v) sucrose substituted for Ficoll during sample denaturation. Heavily stained bands contain approximately 50 ng of RNA.
LANE
Combs 1.25 mm thick were convenient for our purposes. Combs of lucite. which is hydrophilic. formed “hills” around the wells, whereas combs of Teflon, which is hydrophobic. formed “valleys” around the wells. Polyethylene distorted agarose less than other polymers and is the material of choice for making combs. Silver is an especially favorable stain for denatured RNA. In contrast to ethidium bromide, silver stains do not lose sensitivity when nucleic acids are denatured. The staining procedure should be adjusted for changes in gel dimensions. Individual steps must be lengthened for thicker gels and solution volumes should be increased for larger gel volumes. The distilled water washes appear to be critical. Additional washing should be considered wherever staining is poor. The silver stain detected a band containing 300 pg of TMV RNA (data not shown). Successful electrophoresis of RNA requires inactivation of ribonuclease. Denatured (unfolded) RNA is especially vulnerable to ribonuclease. This procedure incorporates iodoacetic acid into the gel matrix and formaldehyde into the electrophoresis buffer to inhibit ribonuclease. Ribonuclease digestion causes RNA bands on gels to smear. The sharp bands of Figs. 1 and 2 show that ribonuclease has been successfully inhibited. This procedure is simple, rapid, and sensitive. Casting gels on a plastic support facilitates silver staining and yields a permanent, convenient record. Glyoxal modification gives accurate molecular weights and even the weaker denaturant. formaldehyde, gives accurate molecular weights for the RNAs we have used. We routinely use the procedure to determine sizes of plant viral RNAs. ACKNOWLEDGMENTS We thank Yukio Shirako for pointing out the virtues ofglyoxal and suggesting that its purification is supertluous. We thank Ken Narva for supplying E. coli.
RNA
137
ELECTROPHORESIS
REFERENCES I. Loening. U. E. (1967) Eioch~. J 102, 25 i-257. 2. Peacock, A. C., and Dingman. C. W. ( 1967) UrochmG.srr~s 6, 18 I& 1827. 3. Loening. U. E. ( 1969) Biochw J. 113, I 13-l 3 I. 4. Dingman, C. W.. Fisher, M. P.. and Kakefuda. T. ( 1973) Eiocher~~i.sfr~* 1 I. 1242- I25O. 5. Staynov. D. Z.. Pinder, J. C.. and Gratrer. W. B. (1972) Nutwe .‘l’rbr Bid 235. 108-l IO. 6. Boedtker. H. ( I97 I ) Riochirx BI~FJJ~I~T :lcto 240, 448-453. 7. Locker, J. (1979) Anal. Bimhern. 98, 358-367. 8. Reijnders. L., Sloof, P.. Sisal. J.. and Borst. P. ( 1973) Biochim Biuph)x .Aclu 324. 320-333. 9. McMaster. G. K.. and Carmichael. G. G. (lY77) Proc .Vut/ tcud. Sc?. L’S.4 74, 4835-4838. IO. Bailey. J. M., and Davidson. N. (1976) .-Id Birtd7er77. 70, 75-85. 1 I. Allen. R. C. (1983) Trcv7d.~.-l~7ul. C’hrt~~ 2. 206-l IO. 12. Willoughby. E. W., and Lambert. A. (1083) .1rrrri Bioc~/7rm. 130, 353-358. 13. Lane. L. C. ( 1986) 777 Methods in Enzymology
14. 15.
16. 17. IX.
(Weissbach. A.. and Weissbach, H., Eds.). Vol. I 18. pp. 687-696, Academic Press. Orlando, FL. Ltttauer. U. Z.. and Eisenberg. H. ( 1959) Riod7im Biqhys. .k!u 32, 320-337. Goelet. P.. Lomonosoff. G. P.. Butler. P. J. G.. Akam. M. E.. Gait. M. J.. and Karn. J. ( 1987) Proc~. hhll. Acad .T~Y. C’S,,-l 79, 58 18-5817. Ahlquist. P.. Dasgupta. R., and Kaesberg. P. (1984) .I. .\f~d Biol. 172. 369-383. Gustafson. CT.. and Armour. S. L. ( I S86) ,~~rr~/~~ir~ .lcrd.s Kr\ 14, 3895-3909. Gustafson. G.. Hunter. B., Hanau. R., Armour, S. L.. and Jackson. 4. 0. (1987) Irro/o,ej~ 158, 394-406.
19. Brosius. J.. Palmer. M. I-.. Kennedy. Noller, H. F. (197X) Proc~. Xarl :lcud 75, 480 I-4805. 20. Brosius. J.. Dull, 7‘. J.. and Noller. H. Pm AKull .hd Sd. l’S.s.-I 77, 701-204. 2 I. Wellaucr. P. K., and Dawid. I. B. ( 1973) :lad. So I:‘? 1 70, 2827-283 I. 17. Lane. L. C. (lY74) Irrolqqy 58. 323-333. 23. Chiko, A. W. (1975) ITrol<1,cj’63, II j-121.
P. J., and S,i. L!S. I F. ( 1980) I’ro<, ,vut/.
BIOCHEMISTRY
169, 132- 137 (I 988)
Agarose Gel Electrophoresis
of Denatured
RNA with Silver Staining
ROSE N. SKOPP AND LESLIE C. LANE Department
of Plant Patholqqy.
Llniversity
qf’Nehrasku.
Lincoln.
Nebraska
68583-07-72
Received June 18. 1987 This paper describes agarose gel electrophoresis and silver staining of denatured RNAs. Glyoxal- or formaldehyde-denatured RNAs are electrophoresed in an agarose gel cast on a plastic support using an inert low conductivity buffer. Following electrophoresis, the gel is stained with a sensitive silver stain. The method produces sharp, well-resolved bands and yields accurate RNA size estimates. Because of its sensitivity and simplicity, it is suitable for routine laboratory use. 8 1988 Academic Press. inc. KEY WORDS: gel electrophoresis; silver stain; RNA: denaturation; molecular weights,
Existing methods for separating denatured RNAs are inconvenient and insufficiently sensitive for routine laboratory use. A review of the literature illustrates the disadvantages of existing methods and suggests improvements. Gel electrophoresis is convenient for separating RNAs (1,2) and estimating their sizes (3). RNA migration rates depend on both molecular weight and conformation (4). Conformational heterogeneity will reduce RNA resolution and conformational differences among RNAs can limit the accuracy of molecular weight estimates. However, denaturants can minimize conformational differences, yielding sharper bands and allowing more accurate size estimates. The denaturants formamide (5) formaldehyde (6) and urea (7.8) reduce heterogeneity but do not completely eliminate conformational differences, whereas glyoxal (9) and methylmercury hydroxide eliminate both conformational differences and heterogeneity. However. these more effective denaturants have disadvantages. Glyoxal-denatured RNAs stain poorly with standard nucleic acid dyes and methylmercury hydroxide is both expensive and toxic. RNA can be electrophoresed in either polyacrylamide or agarose gels. Polyacryl0003-‘697/88
$3.00
CopyrIght t 1988 by Academic Press. Inc. All rights of rcprc&ct,on I” any form reserved
amide gels are relatively unsatisfactory because their pores are too small for large, denatured RNAs. Polyacrylamide is also chemically incompatible with some methods. For example. formaldehyde reacts with polyacrylamide and methylmercury hydroxide inhibits acrylamide polymerization. Agarose is an alternative gel matrix. It can easily be cast on a support medium and it contains large pores. Grades of agarose with high gel strength and low endosmosis are especially suitable for electrophoresis of RNA. Commercial agarose contains ribonuclease which must be inactivated before electrophoresis. Iodoacetate effectively inactivates the ribonuclease in commercial agarose (9). Standard methods for staining denatured RNA are ineffective. Ethidium bromide, the most commonly used stain, detects doublestranded nucleic acids with high sensitivity: however, it stains denatured RNA only faintly. Acridine orange stains denatured RNA (lo), but gives a high background. Silver stains detect double-stranded nucleic acids with high sensitivity (1 1) and in our experience are equally satisfactory for denatured RNA. This paper presents a method for reliable separation and size estimation of RNA dena-
132
RNA ELECTROPHORESIS
tured with formaldehyde or glyoxal. Nucleic acid is separated in agarose cast on GelBond film using an inert, low conductivity electrophoresis buffer containing iodoacetate to inactivate ribonuclease. The silver stain of Willoughby and Lambert (12) detects denatured RNA with high sensitivity. The procedure is simple. rapid, and sensitive. MATERIALS
AND METHODS
Materials GelBond film and low endosmosis (LE)’ high gelling temperature (HGT) agarose were obtained from the FMC Corp. (Rockland. ME). The horizontal gel apparatus was built from lucite with a gel stage of 90 X 110 mm. Reagent grade formaldehyde (Fisher brand, 37%, w/w) containing methanol stabilizer and glyoxal trimeric dihydrate (Sigma, approximately 98%) were used without additional purification. Tungstosilicic acid was purchased from Gallard-Schlesinger Chemical Mfg. Corp. (Carle Place, NY). Other chemicals were reagent grade where available. Methods Viruses were purified by standard procedures (13). Purified preparations were diluted to approximately 0.5 mg/ml with 0.1 M Tris containing 0.05 M HCl. The solution was brought to 3% (w/w) in sodium dodecyl sulfate (SDS). One volume of water saturated phenol was mixed with 1 vol of virus (400-J aliquots) and heated for 5 min at 60°C. The sample was cooled and centrifuged for 5 min at 8OOOg. The upper (aqueous) phase was withdrawn and precipitated with 2-3 vol of reagent grade ethanol for 2 h at -20°C or for 1 h at -80°C. Recovery of the aqueous phase ’ Abbreviations used: LE, low endosmosis; HGT, high gelling temperature: SDS, sodium dodecyl sulfate; DMSO, dimethyl sulfoxide; TMV, tobacco mosaic virus; BMV, brome mosaic virus: BSMV. barley stripe mosaic virus: GC, guanine-cytosine.
133
was about 50% of the initial aqueous volume. The suspension was centrifuged for 10 min at 8OOOg. The supernatant was discarded and the pellet was dried under vacuum. Escherichia coli total nucleic acids were extracted by standard methods ( 14). Formaldeh~~de modijication. Vacuumdried RNA pellets were dissolved in about 100 ~1 of 2% (w/w) SDS, 10% (w/v) sucrose, 2 mM Na,-EDTA, 75 mM N-ethylmorpholine, and 50 mM phosphoric acid. Formaldehyde was added to a final concentration of 3.7% (w/w). The sample was heated for 10 min at 70°C. Small aliquots (3-10 ~1) were electrophoresed. C;lJv.t-al mod$cation. Vacuum-dried RNA pellets were dissolved in approximately 100 ~1 of a solution containing 1 M glyoxal. 75 mM iii-ethylmorpholine, 50 mM phosphoric acid, and 50% (v/v) dimethyl sulfoxide (DMSO). The sample was heated for 10 min at 70°C. Small aliquots (3-10 ~1) were electrophoresed. Both formaldehydeand glyoxal-modified samples may be stored for several months at -30°C . Ckl pwparation. HGT agarose was dissolved by heating in 1%’ (w/v) 75 mM h’-ethylmorpholine, 50 mM phosphoric acid. 0. l?ic (w/w) SDS, and 8 mM iodoacetic acid: 10 ml was poured onto 85 X loo-mm GelBond film. A 1.25-mm-thick polyethylene comb with 4-mm teeth was inserted into the gel and held in place with tape. After the agarose solidified (at least 10 min) the comb was carefully removed. Note that sample wells are difficult to locate once the gel is submerged. One method to facilitate loading is to mark well positions on the back of the plastic support. Prior to electrophoresis, the gel was submerged and incubated for 1 h in electrophoresis buffer (75 mM N-ethylmorpholine, 50 mM phosphoric acid. 1.85% (w/w) formaldehyde). Samples were applied and the gel was electrophoresed at a current flux of about 12 mA/cm’ for 90 min. Silver stain. Following electrophoresis, the gel was stained by the procedure of Wil-
134
SKOPP AND LANE
loughby and Lambert ( 12). The gel was fixed for 15 min in a solution containing 17.3 g of sulfosalicylic acid, 25 g of trichloroacetic acid, 25 g of zinc sulfate, and 500 ml of distilled water. After washing the gel three times for 10 min each in distilled water, it was dehydrated for 5 min in ethanol. The gel was dried with a heat lamp or hot air dryer. The silver stain was prepared from two stock solutions, A and B. Solution A contained 25 g of anhydrous Na2C03 and 500 ml of distilled water. Solution B consisted of 1 g of NH4N03, I g of AgN03, 5 g of tungstosilicic acid, 7.0 ml of 37% (w/w) formaldehyde, and 500 ml of distilled water. To stain, 13.5 ml of solution B was mixed with 6.5 ml of solution A. The dried gel was immersed in the staining bath and vigorously agitated. A white precipitate, which gradually darkened to gray, formed in the solution. When this precipitate became dark gray or when bands first appeared, the gel was removed. rinsed briefly (3-4 s) in distilled water, and placed in fresh staining solution. Generally, two staining cycles were sufficient but lightly loaded gels required three. Staining was stopped by placing the gel in 1WI(v/v) acetic acid for about 5 min. The gel was then rinsed briefly in distilled water and dried by evaporation. RESULTS
Both formaldehydeand glyoxal-denatured RNAs electrophoresed as distinct bands on agarose gels. Figure 1 shows RNAs from tobacco mosaic virus (TMV), brome mosaic virus (BMV), and barley stripe mosaic virus (BSMV) denatured with formaldehyde and glyoxal. Note that glyoxal-modified RNAs migrated slightly slower than formaldehyde-modified counterparts, suggesting that they have more extended configurations. Similarly, Fig. 2 shows the 16 and 23 S 6. coli rRNAs denatured with formaldehyde and glyoxal. Mixtures of BMV and TMV RNAs denatured with formaldehyde or glyoxal served as size standards.
a
b
c
d
FIG. I Formaldehyde- and glyoxal-denatured RNAs electrophoresed on a horizontal 1% agarose gel stained with silver. (a) TMV and BMV RNAs denatured with formaldehyde. (b) TMV (6397 nucleotides) and BMV RNAs (3234, 7865. 21 14. and 876 nucleotides) denatured with glyoxal. (c) BSMV RNAs (3890. 3289, and 279 I nucleotides) denatured with formaldehyde. (d) BSMV RNAs denatured with glyoxal. Heavily stained bands contain approximately 50 ng of RNA.
Both formaldehyde and glyoxal methods gave accurate molecular size estimates. A graph of the logarithm of the molecular size versus mobility gave a straight line. The slope of this line depended on the electrophoretic conditions and differed between formaldehyde and glyoxal-denatured RNAs. Standard curves were constructed using previously established values for the sizes of TMV (6397 nucleotides (15)) and BMV RNAs (3234. 2865. 2114. and 876 nucleotides ( 16)). The molecular sizes of BSMV RNAs 2 and 3, and E. coli 16 and 23 S rRNAs estimated from these curves using regression analysis. were consistent with nucleotide sequences ( 17-20). The correlation coefficients ranged from 0.992 to 0.999. To obtain reliable size estimates, standards and samples must be treated with the same denaturant. Table 1 compares published estimates for RNA molecular sizes with those determined by the glyoxal and formaldehyde methods described here.
RNA a
b
c
135
ELECTROPHORESIS
d
FIG. 2. Formaldehydeand glyoxal-denatured RNAs electrophoresed on a horizontal 1% agarose gel stained with silver. (a) E. co/i rRNAs (2904 and IS41 nucleotides) denatured with formaldehyde (the band closest to the origin is DNA). (b) TMV (6397 nucleotides) and BMV RNAs (3234, 2865, 21 14. and 876 nucleotides) denatured with formaldehyde. (c) E. co/i rRNAs denatured with glyoxal. (d) TMV and BMV RNAs denatured with glyoxal. Heavily stained bands contain approximately 50 ng of RNA.
In early experiments, RNA was denatured with formaldehyde in the presence of Ficoll. However, this produced indistinct, poorly defined RNA bands which smeared toward the origin (Fig. 3). Adding Ficoll after formaldehyde denaturation produced similar patterns. Extended incubation of RNA with formaldehyde and Ficoll progressively increased smearing and retarded migration (data not shown). Replacing Ficoll with 10%) (w/v) sucrose eliminated this problem. Ficoll did not interfere with electrophoresis of glyoxal denatured RNAs.
(21)) suggested that formaldehyde does not completely denature GC-rich RNAs. Since none of the RNAs discussed here were GC rich, it is unwise to assume that formaldehyde will completely denature all RNAs. Nonetheless, this procedure yields accurate molecular weights for BSMV RNAs where earlier formaldehyde methods gave high values (22). Two explanations can be entertained for the inaccuracy of earlier values: reversibility of formaldehyde modification and interaction of formaldehyde-modified RNA with polyacrylamide. Omitting formaldehyde from the running buffer does not influence the apparent size of BSMV RNAs in our system (data not shown). This suggests that polyacrylamide may be an unreliable medium for electrophoresis of formaldehyde-modified RNAs. The apparent crosslinking of RNA to Fico11 by formaldehyde also suggests potentially undesirable aldehyde chemistry. If formaldehyde crosslinks RNA to Ficoll, it likely crosslinks RNA to sucrose. The sucrose adduct would be small and unlikely to influence electrophoretic properties of the RNA.
TABLE COMPARISON
OF PUBLISHED
MOLECULAR WEIGHTS THE GLYOXAL AND
I ESTIMATES
WITH THOSE FORMALDEHYDE
FOR RNA
DETERMINED METHODS
~1’
Sire in nucleotides RNA BSMV- I BSMV-2 BSMV-3 E coli(l6S) E. cdi (23 S)
Glyoxal 3910 3250 2700 l52Ok 3910
+ 210 i I40 + 140 60 f 80
Formaldehyde 3830 3270 2720 15502 2850
r 120 If I30 t 150 90 f 130
Published 3890” 3289(17) 2791 (IX) 1541 (19) 2904 (20)
DISCUSSION
This procedure combines several techniques to give simple, high resolution separation of denatured RNA on horizontal agarose gels. Both glyoxal and formaldehyde derivatives yield accurate molecular weight estimates. Earlier studies (for example, Ref.
Note. RNA sizes were determined from standard curves derived using denatured RNAs of known size. e.g., TMV and BMV. Standards and samples were treated with the same denaturant. Each value represents the average of at least five separate determinations using different gels. ’ Estimate based on the chemical sequence of RNA-3 (17) and particle length ofvirions (23).
136
SKOPP
AND
Nonetheless, crosslinking of nucleic acids to carbohydrates could influence other applications of formaldehyde, Reagents have been chosen for convenience. Both the anion and cation of the buffer have low ionic mobility and are inert to aldehydes. Any buffer which satisfies these criteria should be satisfactory. Both A’-ethylmorpholine and phosphoric acid are liquids and can be dispensed volumetrically. The buffer neutralizes acids which could interfere with RNA modification. If buffering is insufficient, acids can be neutralized by adding more N-ethylmorpholine. In our experience. ion-exchange purification of glyoxal is unnecessary. The thickness and composition of the well-forming comb influence the sharpness of RNA bands. Teflon lucite, and polyethylene combs varying in thickness from 0.75 to 2.5 mm were tried. In general, thinner combs gave sharper bands but wells formed by 0.75-mm combs were difficult to load. a
b
c
FIG. 3. Formaldehyde-denatured RNAs from TMV (6397 nucleotides) and BMV (3234, 2865, 21 14. and 876 nucleotides) electrophoresed on a horizontal 1% agarose gel and stained with silver. (a) 10% (w/v) Ficoll added to the sample after denaturation. (b) 10% (w/v) Ficoll present during sample denaturation. (c) 10% (w/v) sucrose substituted for Ficoll during sample denaturation. Heavily stained bands contain approximately 50 ng of RNA.
LANE
Combs 1.25 mm thick were convenient for our purposes. Combs of lucite. which is hydrophilic. formed “hills” around the wells, whereas combs of Teflon, which is hydrophobic. formed “valleys” around the wells. Polyethylene distorted agarose less than other polymers and is the material of choice for making combs. Silver is an especially favorable stain for denatured RNA. In contrast to ethidium bromide, silver stains do not lose sensitivity when nucleic acids are denatured. The staining procedure should be adjusted for changes in gel dimensions. Individual steps must be lengthened for thicker gels and solution volumes should be increased for larger gel volumes. The distilled water washes appear to be critical. Additional washing should be considered wherever staining is poor. The silver stain detected a band containing 300 pg of TMV RNA (data not shown). Successful electrophoresis of RNA requires inactivation of ribonuclease. Denatured (unfolded) RNA is especially vulnerable to ribonuclease. This procedure incorporates iodoacetic acid into the gel matrix and formaldehyde into the electrophoresis buffer to inhibit ribonuclease. Ribonuclease digestion causes RNA bands on gels to smear. The sharp bands of Figs. 1 and 2 show that ribonuclease has been successfully inhibited. This procedure is simple, rapid, and sensitive. Casting gels on a plastic support facilitates silver staining and yields a permanent, convenient record. Glyoxal modification gives accurate molecular weights and even the weaker denaturant. formaldehyde, gives accurate molecular weights for the RNAs we have used. We routinely use the procedure to determine sizes of plant viral RNAs. ACKNOWLEDGMENTS We thank Yukio Shirako for pointing out the virtues ofglyoxal and suggesting that its purification is supertluous. We thank Ken Narva for supplying E. coli.
RNA
137
ELECTROPHORESIS
REFERENCES I. Loening. U. E. (1967) Eioch~. J 102, 25 i-257. 2. Peacock, A. C., and Dingman. C. W. ( 1967) UrochmG.srr~s 6, 18 I& 1827. 3. Loening. U. E. ( 1969) Biochw J. 113, I 13-l 3 I. 4. Dingman, C. W.. Fisher, M. P.. and Kakefuda. T. ( 1973) Eiocher~~i.sfr~* 1 I. 1242- I25O. 5. Staynov. D. Z.. Pinder, J. C.. and Gratrer. W. B. (1972) Nutwe .‘l’rbr Bid 235. 108-l IO. 6. Boedtker. H. ( I97 I ) Riochirx BI~FJJ~I~T :lcto 240, 448-453. 7. Locker, J. (1979) Anal. Bimhern. 98, 358-367. 8. Reijnders. L., Sloof, P.. Sisal. J.. and Borst. P. ( 1973) Biochim Biuph)x .Aclu 324. 320-333. 9. McMaster. G. K.. and Carmichael. G. G. (lY77) Proc .Vut/ tcud. Sc?. L’S.4 74, 4835-4838. IO. Bailey. J. M., and Davidson. N. (1976) .-Id Birtd7er77. 70, 75-85. 1 I. Allen. R. C. (1983) Trcv7d.~.-l~7ul. C’hrt~~ 2. 206-l IO. 12. Willoughby. E. W., and Lambert. A. (1083) .1rrrri Bioc~/7rm. 130, 353-358. 13. Lane. L. C. ( 1986) 777 Methods in Enzymology
14. 15.
16. 17. IX.
(Weissbach. A.. and Weissbach, H., Eds.). Vol. I 18. pp. 687-696, Academic Press. Orlando, FL. Ltttauer. U. Z.. and Eisenberg. H. ( 1959) Riod7im Biqhys. .k!u 32, 320-337. Goelet. P.. Lomonosoff. G. P.. Butler. P. J. G.. Akam. M. E.. Gait. M. J.. and Karn. J. ( 1987) Proc~. hhll. Acad .T~Y. C’S,,-l 79, 58 18-5817. Ahlquist. P.. Dasgupta. R., and Kaesberg. P. (1984) .I. .\f~d Biol. 172. 369-383. Gustafson. CT.. and Armour. S. L. ( I S86) ,~~rr~/~~ir~ .lcrd.s Kr\ 14, 3895-3909. Gustafson. G.. Hunter. B., Hanau. R., Armour, S. L.. and Jackson. 4. 0. (1987) Irro/o,ej~ 158, 394-406.
19. Brosius. J.. Palmer. M. I-.. Kennedy. Noller, H. F. (197X) Proc~. Xarl :lcud 75, 480 I-4805. 20. Brosius. J.. Dull, 7‘. J.. and Noller. H. Pm AKull .hd Sd. l’S.s.-I 77, 701-204. 2 I. Wellaucr. P. K., and Dawid. I. B. ( 1973) :lad. So I:‘? 1 70, 2827-283 I. 17. Lane. L. C. (lY74) Irrolqqy 58. 323-333. 23. Chiko, A. W. (1975) ITrol<1,cj’63, II j-121.
P. J., and S,i. L!S. I F. ( 1980) I’ro<, ,vut/.