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An improved method of detecting DNA-RNA hybrids bound to nitrocellulose filters
ANALYTICAL
An
37, 330-332 (1970)
BIOCHEMISTRY
improved
Method Bound
J. C. DANIEL, Department
of
Detecting
to Nitrocellulose
DNA-RNA
Hybrids
Filters’
R. F. GREENE, R. A. MITCHELL, R. A. FLICKINGER
of Biology, State University of New Buffalo, New York 14214
AND
York at Buffalo,
Received February 18, 1970 The nitrocellulose filter technique of Gillespie and Spiegelman (1) has been widely used for the detection of DNA-RNA hybrids. This method allows samples to be easily handled and prevents interference by DNA-DNA interactions. Assay of hybrids presents serious difficulties when tritium-labeled RNA of low specific activity is used. The ability of nitrocellulose filters to absorb beta particles, as well as the selfabsorption by the H3-RNA-DNA hybrids, makes the accurate determination of radioactivity impossible when counting in the liquid scintillation system. The present paper describes an improved method for the assay of these hybrids. METHODS
DNA fikers were prepared and cut to a diameter of 6 mm, each filter containing 5 pug of heat-denatured Rana pipiens red blood cell DNA. Two experimental filters and one control filter, not containing DNA, were placed in 9 X 30 mm shell vials. RNA, which had been extracted from Rana pipiens tailbud embryos was tritium-labeled with dimethyl sulfate (2)) and was dissolved in 2 x SSC,2 30% formamide, and 0.15 ml of this solution was added ,to each vial. The vials were incubated at 25°C for 50 hours. The filters were removed, washed in 2 X SSC at 25”, treated with RNase (20 pg/ml) at 25” for 45 minutes to remove unhybridized RNA, washed again with 2 X SSC, then air-dried and placed in separate scintillation bottles. Then 10 ml of toluene-POPPOPOP scintillation cocktail was added and the samples were counted for 20 minutes each. After they had been counted, the filters were removed ‘This research wa.~ supported by NIH grant GM 1623601 and NSF grant GB 5500. ‘SSC is 0.15 M NaCl f 0.015 M sodium citrate. 330
DETECTION
OF DNA-RNA
331
HYBRIDS
from the bottles, air-dried; and placed in new scintillation bottles. The hybrids were hydrolyzed from the filters by the addition to each bottle of 0.3 ml of a solution containing 500 pg pancreatic RNase A per milliliter and 500 ,pg crude DNase 1 per milliliter in 0.01 M Tris, pH 7.5, 2 mM MgCl,. The use of DNase was necessary to remove the DNA-RNA hybrid from the filters and RNase was used to digest the RNA so that it would not precipitate in the scintillation cocktail. After the bottles were incubated at 37” for 8 hours, 10 ml of mtoluene cocktail containing 7% (v/v) BIOSOLV solubilizer (Beckman) was added and the samples were recounted. RESULTS
AND
DISCUSSION
A typical experiment in which increasing concentrations of H3-RNA were incubated with filters containing 5 pg of DNA is shown in Table 1. The counts per minute detected were Jtwice as high after hydrolysis than before hydrolysis. The average deviation from the mean for duplicates in this experiment and four other experiments of this type wa#s 9.5% without enzymic treatment, but. only 4.3% after hydrolysis. The reason for the lower counts before hydrolysis can be ascribed to the ability of the nitrocellulose filters to absorb beta particles and also to self-absorption by the DNA-RNA hybrids. Hydrolysis of the hybrids from the filters appears to be complete, since after hydrolysis experimental filters were found to retain only 0.003 to 0.0040/o of the total input counts, which is the same level as was retained on blank Counts per Minute of Frog Cell DNA on Nitrocellulose
Amt.
of input (crdml)
TABLE 1 Tailbud Embryo H3-RN 4 Hybridized to 5 rg Frog Red Blood Filters before and after D’gestion of Duplexes by RNase and DNase (500pg/ml Each)O
RNA
Total cpm before hydrolysis
31
l-5.1 10.0 20.6 14.1 25.4 27.6 33.1 53.6 71.5 63.3 100.2 91.6
63 125 250 500 1,000
y The
levels
of cpm
Total cpm after hydrolysis
of the control
filters
were
41.8 40.7 47.6 55.2 71.4 66.1 103.4 93.9 140.5 140.1 221.9 208.1 subtracted
from
each
value.
332
DANIEL
ET AL.
filters that did not contain DNA. This low level is probably caused by contamination from protein that became labeled during the methylation procedure. When RNA labeled in vivo wi’th H3-uridine w.as used in other experiments, no detectable counts were retained on blank filters or on filters containing DNA. In addition, DNA determinations on filters containing 30 and 133 fig of denatured DNA showed that after hydrolysis with DNase (500 ,&ml), no detectable DNA remained on the filters. In these experiments RNA with a specific activity of 800 cpm/pg was used. Similar results were obtained when RNA labeled in vitro with dimethyl sulf.ate having a specific activity of 5000 cpm/pg was used and when H3-uridine in viva labeled RNA with a specific activity of 6000 cpm/pg was used. In order to determine specific aotivities of RNA sa#mples accurately it was found necessary to digest the RNA before counting. Aliquots of H3-RNA pipetted directly into the scintillation cocktail containing the solubilizer gave levels of radioactivity approximately 30% lower than that obtained when identical aliquots were first hydrolyzed and then counted. The level of detectable counts decrease in unhydrolyzed RNA samples with time and this decrease can be partially reversed by shaking the vials. This suggests that there is a gradual precipitation of the unhydrolyzed RNA in the scintillation cocktail resulting in increased self-absorption of counts. RNA samples and DNA-RNA hybrids that had been hydrolyzed by RNase and DNase showed no detectable loss of counts with time. Since the absorption by nitrocellulose filters and self-absorption by the DNA-RNA hybrids are eliminated by digestion of the filters, quenching of the sample can be accurately measured by the scintillation counter and disintegrations per minute precisely calculated from a counting efficiency curve. SUMMARY
The enzymic removal of DNA-RNA hybrids from nitrocellulose filters before counting results in an increase in the detectable counts per minute and a decrease in the variability between duplicate samples. This permits the use of filters containing smaller amounts of DNA, and the use of RNA which has a lower specific activity, without losing statistical accuracy. Both accuracy and precision in the determination of specific activities of RNA samples are increased by digesting the RNA prior to counting. REFERENCES 1. GILLESPIE, D., AND SPIECELMAN, S., J. Mol. Bid. 2. SMITH, K. D.. ARMSTRONG, J. L., AND MCCARTHY, 142, 323 (1967).
12, 829 (1965). B. J., Biochim. Biophys. Acta
An
37, 330-332 (1970)
BIOCHEMISTRY
improved
Method Bound
J. C. DANIEL, Department
of
Detecting
to Nitrocellulose
DNA-RNA
Hybrids
Filters’
R. F. GREENE, R. A. MITCHELL, R. A. FLICKINGER
of Biology, State University of New Buffalo, New York 14214
AND
York at Buffalo,
Received February 18, 1970 The nitrocellulose filter technique of Gillespie and Spiegelman (1) has been widely used for the detection of DNA-RNA hybrids. This method allows samples to be easily handled and prevents interference by DNA-DNA interactions. Assay of hybrids presents serious difficulties when tritium-labeled RNA of low specific activity is used. The ability of nitrocellulose filters to absorb beta particles, as well as the selfabsorption by the H3-RNA-DNA hybrids, makes the accurate determination of radioactivity impossible when counting in the liquid scintillation system. The present paper describes an improved method for the assay of these hybrids. METHODS
DNA fikers were prepared and cut to a diameter of 6 mm, each filter containing 5 pug of heat-denatured Rana pipiens red blood cell DNA. Two experimental filters and one control filter, not containing DNA, were placed in 9 X 30 mm shell vials. RNA, which had been extracted from Rana pipiens tailbud embryos was tritium-labeled with dimethyl sulfate (2)) and was dissolved in 2 x SSC,2 30% formamide, and 0.15 ml of this solution was added ,to each vial. The vials were incubated at 25°C for 50 hours. The filters were removed, washed in 2 X SSC at 25”, treated with RNase (20 pg/ml) at 25” for 45 minutes to remove unhybridized RNA, washed again with 2 X SSC, then air-dried and placed in separate scintillation bottles. Then 10 ml of toluene-POPPOPOP scintillation cocktail was added and the samples were counted for 20 minutes each. After they had been counted, the filters were removed ‘This research wa.~ supported by NIH grant GM 1623601 and NSF grant GB 5500. ‘SSC is 0.15 M NaCl f 0.015 M sodium citrate. 330
DETECTION
OF DNA-RNA
331
HYBRIDS
from the bottles, air-dried; and placed in new scintillation bottles. The hybrids were hydrolyzed from the filters by the addition to each bottle of 0.3 ml of a solution containing 500 pg pancreatic RNase A per milliliter and 500 ,pg crude DNase 1 per milliliter in 0.01 M Tris, pH 7.5, 2 mM MgCl,. The use of DNase was necessary to remove the DNA-RNA hybrid from the filters and RNase was used to digest the RNA so that it would not precipitate in the scintillation cocktail. After the bottles were incubated at 37” for 8 hours, 10 ml of mtoluene cocktail containing 7% (v/v) BIOSOLV solubilizer (Beckman) was added and the samples were recounted. RESULTS
AND
DISCUSSION
A typical experiment in which increasing concentrations of H3-RNA were incubated with filters containing 5 pg of DNA is shown in Table 1. The counts per minute detected were Jtwice as high after hydrolysis than before hydrolysis. The average deviation from the mean for duplicates in this experiment and four other experiments of this type wa#s 9.5% without enzymic treatment, but. only 4.3% after hydrolysis. The reason for the lower counts before hydrolysis can be ascribed to the ability of the nitrocellulose filters to absorb beta particles and also to self-absorption by the DNA-RNA hybrids. Hydrolysis of the hybrids from the filters appears to be complete, since after hydrolysis experimental filters were found to retain only 0.003 to 0.0040/o of the total input counts, which is the same level as was retained on blank Counts per Minute of Frog Cell DNA on Nitrocellulose
Amt.
of input (crdml)
TABLE 1 Tailbud Embryo H3-RN 4 Hybridized to 5 rg Frog Red Blood Filters before and after D’gestion of Duplexes by RNase and DNase (500pg/ml Each)O
RNA
Total cpm before hydrolysis
31
l-5.1 10.0 20.6 14.1 25.4 27.6 33.1 53.6 71.5 63.3 100.2 91.6
63 125 250 500 1,000
y The
levels
of cpm
Total cpm after hydrolysis
of the control
filters
were
41.8 40.7 47.6 55.2 71.4 66.1 103.4 93.9 140.5 140.1 221.9 208.1 subtracted
from
each
value.
332
DANIEL
ET AL.
filters that did not contain DNA. This low level is probably caused by contamination from protein that became labeled during the methylation procedure. When RNA labeled in vivo wi’th H3-uridine w.as used in other experiments, no detectable counts were retained on blank filters or on filters containing DNA. In addition, DNA determinations on filters containing 30 and 133 fig of denatured DNA showed that after hydrolysis with DNase (500 ,&ml), no detectable DNA remained on the filters. In these experiments RNA with a specific activity of 800 cpm/pg was used. Similar results were obtained when RNA labeled in vitro with dimethyl sulf.ate having a specific activity of 5000 cpm/pg was used and when H3-uridine in viva labeled RNA with a specific activity of 6000 cpm/pg was used. In order to determine specific aotivities of RNA sa#mples accurately it was found necessary to digest the RNA before counting. Aliquots of H3-RNA pipetted directly into the scintillation cocktail containing the solubilizer gave levels of radioactivity approximately 30% lower than that obtained when identical aliquots were first hydrolyzed and then counted. The level of detectable counts decrease in unhydrolyzed RNA samples with time and this decrease can be partially reversed by shaking the vials. This suggests that there is a gradual precipitation of the unhydrolyzed RNA in the scintillation cocktail resulting in increased self-absorption of counts. RNA samples and DNA-RNA hybrids that had been hydrolyzed by RNase and DNase showed no detectable loss of counts with time. Since the absorption by nitrocellulose filters and self-absorption by the DNA-RNA hybrids are eliminated by digestion of the filters, quenching of the sample can be accurately measured by the scintillation counter and disintegrations per minute precisely calculated from a counting efficiency curve. SUMMARY
The enzymic removal of DNA-RNA hybrids from nitrocellulose filters before counting results in an increase in the detectable counts per minute and a decrease in the variability between duplicate samples. This permits the use of filters containing smaller amounts of DNA, and the use of RNA which has a lower specific activity, without losing statistical accuracy. Both accuracy and precision in the determination of specific activities of RNA samples are increased by digesting the RNA prior to counting. REFERENCES 1. GILLESPIE, D., AND SPIECELMAN, S., J. Mol. Bid. 2. SMITH, K. D.. ARMSTRONG, J. L., AND MCCARTHY, 142, 323 (1967).
12, 829 (1965). B. J., Biochim. Biophys. Acta