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Effect of sucrose gradient composition on resolution of RNA species
ANALYTICAL
45, 271-276 (1972)
BIOCHEMISTRY
Effect
of Sucrose
Gradient
on Resolution MICHAEL Biology
Department,
of RNA
W. NEAL Syracuse
AND
University,
Composition Species
JAMES
R. FLORINI
Syracuse,
New
York
13??10
Received June 11, 1971
Sucrose gradients have been used for many years for the fractionation of RNA. It was believed at first that t,he major function of the gradient was to provide a stable liquid column that would be resistant to thermal or mechanical mixing, and secondarily to provide a more constant sedimentation velocity by increasing frictional drag while increasing centrifugal force as the molecule moves down the tube (1). Later No11 showed that the sedimentation rate was not constant, but that viscous drag increased faster than centrifugal force, causing the molecules to slow down as they moved farther from the center of rotation (2). No11 devised convex exponential gradients to maintain constant sedimentation velocity, and with these gradients obtained an improvement in resolution when analyzing ribosomes. This method has since been applied to RNA using 10-35s convex exponential gradients (3). We have studied the effects of the steepnessof linear sucrose gradients on the fractionation of chicken liver RNA. The resolving power of sucrose gradient’s is strikingly increased as the steepnessof the gradient is increased. The steepest gradient t’hat was used, 10-7070, showed four different species of RNA in the region of the 18s ribosomal peak. Indeed, the resolution obtained from steep gradients is comparable to t’hat observed on gel electrophoresis. In our experience the best resolution is obtained when sucrose concentration and thus viscosity increase rapidly as in the 10-7070 gradients. METHODS
A five week old chicken was sacrificed by decapitation and the liver removed quickly and placed on ice. The RNA was purified by phenol extraction at room temperature by a modification of the procedures of Loeb and Gelboin (4). The homogenizing medium contained 0.1% v/v diethyloxydoformate as an RNase inhibitor. RNA obtained from this procedure has a 260/280 absorbance ratio of 1.9. The RNA was stored as an ethanol precipitate at -10” in 1.0 mg aliquots until used. 271 @ 1972 by Academic
Press. Inc.
272
NEAL
AND
FLORINI
Linear sucrose gradients were prepared at the concentrations indicated; all contained 0.01 M sodium acetate, pH 5.2. The RNA was collected by centrifugation from the ethanol precipitate and dried under nitrogen to remove all traces of ethanol, and then dissolved in 1.0 ml of 0.15 M NaCl. The sample (0.1 ml containing 0.08 mg RNA) was layered on top of 14 ml gradients. The gradients were centrifuged at 4” and 40,000 rpm in the SB-283 rotor of an International B-60 ultracentrifuge for the times indicated. Gradients were fractionated on an ISCO 180 density gradient fractionator, and continuously scanned at 254 nm with an ISCO UA-2 ultraviolet analyzer. Electrophoresis of RNA was done in composite 2.6% acrylamide/0.5% agarose gels using a modification of the procedures of Peacock and Dingman (5) and Loening (6). A 0.04.M Tris/O.O2 M sodium acetate and 2 mM EDTA, pH 7.8, buffer was used in the electrophoresis. The 0.7 X 9.5 cm gels were prerun for 30 min and then approximately 50 pg of RNA in 0.1 ml of a solution containing electrophoresis buffer and 35% sucrose was layered on the top of the gel. The gels were run at 2.5 mA/gel for 5.5 hr at 4”. RESULTS
Figure 1 shows identical samples of chicken liver RNA sedimented through sucrose gradients of varying steepness. The time of centrifugation was adjusted for each gradient to allow the 28s peak to migrate approximately to the same position in the centrifuge tube. As the steepness of the gradient was increased, the bands, particularly for material larger than 5S, became much sharper and distinct peaks appeared in the 10-70s gradient where only shoulders existed in the 10-20s gradient. It was established that the heterogeneity observed in the steeper gradients was not due to discontinuities in the gradient by measuring the refractive index of 1.0 ml samples of the gradients and demonstrating that there was no detectable deviation from linearity. The progression of banding patterns in Fig. 1 demonstrate that the composition of the sucrose gradient had a substantial effect on the resolution of RNA species. After treatment with RNase, all the 254 nm absorbing material remained at the top of a 10-50s sucrose gradient (Fig. 2). To further demonstrate that the heterogeneity observed on the steeper gradients was due to RNA, the sample was treated with 3 M sodium acetate to precipitate rRNA and leave tRNA and DNA in solution (7). The precipitate was redissolved and analyzed on a l&50% sucrose gradient (Fig. 2). The number and proportions of bands are the same as in Fig. 1 except for the expected removal of the 4s tRNA band. These data
RNA
IN
STEEP
SUCROSE
273
GRADIENTS
.8 -
.6 -
VT
DVI
POSITION
I”,
IN GRADIENT
1. Effect of sucrose gradient composition on resolution of chicken liver RNA. Approximately 80 pg chicken liver RNA in 0.1 ml was layered on linear sucrose gradients containing 0.01 M sodium acetate at pH 5.2. Gradients were centrifuged at 40,000 rpm for 8 hr (l&20% gradient), for 11 hr (1~35% gradient), for 14 hr (1050% gradient), or 19.5 hr (lO-70%). For clarity, baselines are displaced serially upward to facilitate direct comparisons of the individual gradients; baseline for each gradient is indicated by the flat region at the right of the figure. FIG.
demonstrate that all the material detected on these gradients was RNA. To ascertain that the apparent heterogeneity in the 18s region was not due to artifacts imposed by the higher concentrations of sucrose in the gradients, a 50 pg sample of RNA was fractionated by composite agarose/acrylamide disc gel electrophoresis (Fig. 3). The pattern observed after staining the gel was similar to that observed with the steeper sucrose gradients. It should be noted, however, that on the gels RNA’s
274
NEAL
AND
FLORINI
I
TOP
BOTTOF POSITION IN GRADIENT
FIG. 2. Effect of precipitation with sodium acetate and treatment with RNase on sucrose gradient profile of chicken liver RNA: (A) RNA was precipitated overnight at 4” in 3 M sodium acetate, pH 5.6. After collection the RNA was taken up in 0.15 M NaCl and 80 pg was layered on a lo-50% gradient and centrifuged for 14 hr at 40,000 rpm. (B) To 1.0 ml of a solution of RNA (0.6 mg/ml) was added 0.2 ml of a 1.0 mg/ml solution of RNase (any DNase present in the P-L Biochemicals bovine pancreatic RNase was inactivated by prior incubation of the enzyme at 90” for 10 min). On a 1050% sucrose gradient 0.1 ml of this solution was layered and centrifuged for 14 hr at 40,000 rpm at 4”.
larger than 28s do not appear, probably due to the small amount of sample added, or their exclusion from the gel pores. We suggest that the fractionation observed on the steeper gradient is an accurate indication of the t.rue heterogeneity of the RNA sample from chicken liver. Similar preparations from rat liver exhibited only one major peak in the 18X region. DISCUSSION
In comparing sucrose gradients of RNA preparations we have demonstrated a relationship between the steepness of the gradient and degree of resolution. The steeper gradients showed distinct peaks appearing where only shoulders existed in the 10-20s gradient, and there was less
RNA
IN
STEEP
SUCROSE
GRADIEKTS
275
CM FROM ORIGIN 3. Analysis of chicken liver RNA by disc electrophoresis in 2.6% acrylamide/Od% agarose composite gels. Approximately 50 pg RNA in 35% sucrose was layered on the gels and electrophoresed for 5.5 hr at 2.5 mA/gel at 4”. The gels were stained with methylene blue and scanned at 600 nm using a Gilford model 2410 linear transport in a Gilford 240 spectrophotometer. FIG.
diffusion of the bands, particularly for the larger species of RNA, even though the steeper gradients were centrifuged for substantially longer times. It would seem that still steeper gradients might further facilitate fractionation, but there are many technical problems in making and analyzing steeper than l&70% gradients because of the high viscosity of the concentrated sucrose solution. We have made no attempt in this work to determine the biological significance of the heterogeneity observed in the steeper gradients, but have attempted only to determine the conditions that will give the best resolution. However, it was determined that the heterogeneity was not due simply to contamination by DNA or protein in our preparation by showing that all the material on the gradient was sensitive to DNase-free RNase and precipitated by 3 M sodium acetate. There are several advantages to analyzing RNA on sucrose gradients. Large quantities of material, up to 1.0 mg, can be analyzed using rotors such as t,he International SB 110, while rotors such as the SB 283 can be used to analyze quantities of less than 50 pg. As long as an appropri-
276
NEAL
AND
FLORINI
ate gradient is used, the resolving power of this technique does not depend on the quantity of RNA analyzed. Sucrose gradients can be separated into fractions easily allowing recovery of material for further analysis. In the past, these advantages have been weighed against the disadvantage of relatively poor resolution in shallow sucrose gradients. This disadvantage can be largely overcome by using the steeper gradients to obtain resolution equivalent to that obtained by gel electrophoresis. However, sucrose gradients have some disadvantages that the steeper gradients do not overcome. The time necessary to achieve separation can be quite long; in l&-70$% gradients 18-20 hr of centrifugation was required. Even after this length of time the small RNA’s, 4s and 53, showed no significant separation, For the analysis of these species of RNA, gel electrophoresis is much superior. SUMMARY
Resolution of RNA samples on sucrose gradients was strikingly dependent upon the sucrose concentration in the gradients. Linear 10-70s gradients separated peaks which were barely discernible as shoulders in lO-20% gradients. The resolution obtained in steep sucrose gradients was comparable to that observed upon electrophoresis in acrylamide/ agarose gels. ACKNOWLEDGMENTS We are grateful to David N. Lisi for his instruction in methodology for preparation and analysis of RNA. This work was supported by grants from the Muscular Dystrophy Associations of America and the National Heart and Lung Institute, USPHS.
1. 2. 3. 4. 5. 6.
7.
REFERENCES BRITTEN, R. J., AND ROBERTS, R. B., Science 131, 32 (1960). NOLL, H., Nature 215, 360 (1967). NOLL, H., AND STUTZ, E., in “Methods in Enzymology,” Vol. XII, p. 129. Academic Press, New York/London, 1968. LOEB, L. A., AND GELBOIN, H. V., Proc. Nut. Acad. Sci. U. S. 52, 1219 (1964). PEACOCK, C., AND DINQMAN, C., Biochemistry 7, 668 (1968). LOENING, U. E., Biochem. J. 102, 251 (1967). KIRBY, K. S., Biochem. J. 96, 266 (1965).
45, 271-276 (1972)
BIOCHEMISTRY
Effect
of Sucrose
Gradient
on Resolution MICHAEL Biology
Department,
of RNA
W. NEAL Syracuse
AND
University,
Composition Species
JAMES
R. FLORINI
Syracuse,
New
York
13??10
Received June 11, 1971
Sucrose gradients have been used for many years for the fractionation of RNA. It was believed at first that t,he major function of the gradient was to provide a stable liquid column that would be resistant to thermal or mechanical mixing, and secondarily to provide a more constant sedimentation velocity by increasing frictional drag while increasing centrifugal force as the molecule moves down the tube (1). Later No11 showed that the sedimentation rate was not constant, but that viscous drag increased faster than centrifugal force, causing the molecules to slow down as they moved farther from the center of rotation (2). No11 devised convex exponential gradients to maintain constant sedimentation velocity, and with these gradients obtained an improvement in resolution when analyzing ribosomes. This method has since been applied to RNA using 10-35s convex exponential gradients (3). We have studied the effects of the steepnessof linear sucrose gradients on the fractionation of chicken liver RNA. The resolving power of sucrose gradient’s is strikingly increased as the steepnessof the gradient is increased. The steepest gradient t’hat was used, 10-7070, showed four different species of RNA in the region of the 18s ribosomal peak. Indeed, the resolution obtained from steep gradients is comparable to t’hat observed on gel electrophoresis. In our experience the best resolution is obtained when sucrose concentration and thus viscosity increase rapidly as in the 10-7070 gradients. METHODS
A five week old chicken was sacrificed by decapitation and the liver removed quickly and placed on ice. The RNA was purified by phenol extraction at room temperature by a modification of the procedures of Loeb and Gelboin (4). The homogenizing medium contained 0.1% v/v diethyloxydoformate as an RNase inhibitor. RNA obtained from this procedure has a 260/280 absorbance ratio of 1.9. The RNA was stored as an ethanol precipitate at -10” in 1.0 mg aliquots until used. 271 @ 1972 by Academic
Press. Inc.
272
NEAL
AND
FLORINI
Linear sucrose gradients were prepared at the concentrations indicated; all contained 0.01 M sodium acetate, pH 5.2. The RNA was collected by centrifugation from the ethanol precipitate and dried under nitrogen to remove all traces of ethanol, and then dissolved in 1.0 ml of 0.15 M NaCl. The sample (0.1 ml containing 0.08 mg RNA) was layered on top of 14 ml gradients. The gradients were centrifuged at 4” and 40,000 rpm in the SB-283 rotor of an International B-60 ultracentrifuge for the times indicated. Gradients were fractionated on an ISCO 180 density gradient fractionator, and continuously scanned at 254 nm with an ISCO UA-2 ultraviolet analyzer. Electrophoresis of RNA was done in composite 2.6% acrylamide/0.5% agarose gels using a modification of the procedures of Peacock and Dingman (5) and Loening (6). A 0.04.M Tris/O.O2 M sodium acetate and 2 mM EDTA, pH 7.8, buffer was used in the electrophoresis. The 0.7 X 9.5 cm gels were prerun for 30 min and then approximately 50 pg of RNA in 0.1 ml of a solution containing electrophoresis buffer and 35% sucrose was layered on the top of the gel. The gels were run at 2.5 mA/gel for 5.5 hr at 4”. RESULTS
Figure 1 shows identical samples of chicken liver RNA sedimented through sucrose gradients of varying steepness. The time of centrifugation was adjusted for each gradient to allow the 28s peak to migrate approximately to the same position in the centrifuge tube. As the steepness of the gradient was increased, the bands, particularly for material larger than 5S, became much sharper and distinct peaks appeared in the 10-70s gradient where only shoulders existed in the 10-20s gradient. It was established that the heterogeneity observed in the steeper gradients was not due to discontinuities in the gradient by measuring the refractive index of 1.0 ml samples of the gradients and demonstrating that there was no detectable deviation from linearity. The progression of banding patterns in Fig. 1 demonstrate that the composition of the sucrose gradient had a substantial effect on the resolution of RNA species. After treatment with RNase, all the 254 nm absorbing material remained at the top of a 10-50s sucrose gradient (Fig. 2). To further demonstrate that the heterogeneity observed on the steeper gradients was due to RNA, the sample was treated with 3 M sodium acetate to precipitate rRNA and leave tRNA and DNA in solution (7). The precipitate was redissolved and analyzed on a l&50% sucrose gradient (Fig. 2). The number and proportions of bands are the same as in Fig. 1 except for the expected removal of the 4s tRNA band. These data
RNA
IN
STEEP
SUCROSE
273
GRADIENTS
.8 -
.6 -
VT
DVI
POSITION
I”,
IN GRADIENT
1. Effect of sucrose gradient composition on resolution of chicken liver RNA. Approximately 80 pg chicken liver RNA in 0.1 ml was layered on linear sucrose gradients containing 0.01 M sodium acetate at pH 5.2. Gradients were centrifuged at 40,000 rpm for 8 hr (l&20% gradient), for 11 hr (1~35% gradient), for 14 hr (1050% gradient), or 19.5 hr (lO-70%). For clarity, baselines are displaced serially upward to facilitate direct comparisons of the individual gradients; baseline for each gradient is indicated by the flat region at the right of the figure. FIG.
demonstrate that all the material detected on these gradients was RNA. To ascertain that the apparent heterogeneity in the 18s region was not due to artifacts imposed by the higher concentrations of sucrose in the gradients, a 50 pg sample of RNA was fractionated by composite agarose/acrylamide disc gel electrophoresis (Fig. 3). The pattern observed after staining the gel was similar to that observed with the steeper sucrose gradients. It should be noted, however, that on the gels RNA’s
274
NEAL
AND
FLORINI
I
TOP
BOTTOF POSITION IN GRADIENT
FIG. 2. Effect of precipitation with sodium acetate and treatment with RNase on sucrose gradient profile of chicken liver RNA: (A) RNA was precipitated overnight at 4” in 3 M sodium acetate, pH 5.6. After collection the RNA was taken up in 0.15 M NaCl and 80 pg was layered on a lo-50% gradient and centrifuged for 14 hr at 40,000 rpm. (B) To 1.0 ml of a solution of RNA (0.6 mg/ml) was added 0.2 ml of a 1.0 mg/ml solution of RNase (any DNase present in the P-L Biochemicals bovine pancreatic RNase was inactivated by prior incubation of the enzyme at 90” for 10 min). On a 1050% sucrose gradient 0.1 ml of this solution was layered and centrifuged for 14 hr at 40,000 rpm at 4”.
larger than 28s do not appear, probably due to the small amount of sample added, or their exclusion from the gel pores. We suggest that the fractionation observed on the steeper gradient is an accurate indication of the t.rue heterogeneity of the RNA sample from chicken liver. Similar preparations from rat liver exhibited only one major peak in the 18X region. DISCUSSION
In comparing sucrose gradients of RNA preparations we have demonstrated a relationship between the steepness of the gradient and degree of resolution. The steeper gradients showed distinct peaks appearing where only shoulders existed in the 10-20s gradient, and there was less
RNA
IN
STEEP
SUCROSE
GRADIEKTS
275
CM FROM ORIGIN 3. Analysis of chicken liver RNA by disc electrophoresis in 2.6% acrylamide/Od% agarose composite gels. Approximately 50 pg RNA in 35% sucrose was layered on the gels and electrophoresed for 5.5 hr at 2.5 mA/gel at 4”. The gels were stained with methylene blue and scanned at 600 nm using a Gilford model 2410 linear transport in a Gilford 240 spectrophotometer. FIG.
diffusion of the bands, particularly for the larger species of RNA, even though the steeper gradients were centrifuged for substantially longer times. It would seem that still steeper gradients might further facilitate fractionation, but there are many technical problems in making and analyzing steeper than l&70% gradients because of the high viscosity of the concentrated sucrose solution. We have made no attempt in this work to determine the biological significance of the heterogeneity observed in the steeper gradients, but have attempted only to determine the conditions that will give the best resolution. However, it was determined that the heterogeneity was not due simply to contamination by DNA or protein in our preparation by showing that all the material on the gradient was sensitive to DNase-free RNase and precipitated by 3 M sodium acetate. There are several advantages to analyzing RNA on sucrose gradients. Large quantities of material, up to 1.0 mg, can be analyzed using rotors such as t,he International SB 110, while rotors such as the SB 283 can be used to analyze quantities of less than 50 pg. As long as an appropri-
276
NEAL
AND
FLORINI
ate gradient is used, the resolving power of this technique does not depend on the quantity of RNA analyzed. Sucrose gradients can be separated into fractions easily allowing recovery of material for further analysis. In the past, these advantages have been weighed against the disadvantage of relatively poor resolution in shallow sucrose gradients. This disadvantage can be largely overcome by using the steeper gradients to obtain resolution equivalent to that obtained by gel electrophoresis. However, sucrose gradients have some disadvantages that the steeper gradients do not overcome. The time necessary to achieve separation can be quite long; in l&-70$% gradients 18-20 hr of centrifugation was required. Even after this length of time the small RNA’s, 4s and 53, showed no significant separation, For the analysis of these species of RNA, gel electrophoresis is much superior. SUMMARY
Resolution of RNA samples on sucrose gradients was strikingly dependent upon the sucrose concentration in the gradients. Linear 10-70s gradients separated peaks which were barely discernible as shoulders in lO-20% gradients. The resolution obtained in steep sucrose gradients was comparable to that observed upon electrophoresis in acrylamide/ agarose gels. ACKNOWLEDGMENTS We are grateful to David N. Lisi for his instruction in methodology for preparation and analysis of RNA. This work was supported by grants from the Muscular Dystrophy Associations of America and the National Heart and Lung Institute, USPHS.
1. 2. 3. 4. 5. 6.
7.
REFERENCES BRITTEN, R. J., AND ROBERTS, R. B., Science 131, 32 (1960). NOLL, H., Nature 215, 360 (1967). NOLL, H., AND STUTZ, E., in “Methods in Enzymology,” Vol. XII, p. 129. Academic Press, New York/London, 1968. LOEB, L. A., AND GELBOIN, H. V., Proc. Nut. Acad. Sci. U. S. 52, 1219 (1964). PEACOCK, C., AND DINQMAN, C., Biochemistry 7, 668 (1968). LOENING, U. E., Biochem. J. 102, 251 (1967). KIRBY, K. S., Biochem. J. 96, 266 (1965).