Improved methods for determination of RNA and DNA

ANALYTICAL

BIOCHMISTRY

Improved

Methods

31, 42-60 (1969)

for Determination

of RNA and DNA’

D. W. HATCHER Analytical

Chemistry

AND GERALD GOLDSTEIN Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 Received January

29, 1969

The Oak Ridge National Laboratory is currently processing Escherichia coli cells for the purpose of isolating gram quantities of various ribonucleic acids in pure form. In attempting to monitor the efficiency of the separations employed, Iwe found that the conventional orcinol calorimetric methods for RNA (1) and diphenylamine calorimetric method for DNA (1,2) are both subject to serious interference by various salts, buffers, and other reagents used in the separations. Some of these interferences could be removed by extensive dialysis while others apparently associate with nucleic acids and could not be removed. Attempts to isolate RNA and DNA by acid precipitation schemes (1,3,4) were also unsuccessful. We present here a technique for precipitating nucleic acids as their Cd (II) salts and modifications of the orcinol and diphenylamine methods which minimize the effects of interferences and greatly improve the reliability of these methods when applied to complex mixtures. MATERIALS

Orcinol (&methylresorcinol, Matheson, Coleman & Bell), recrystallized from benzene; 2 gm dissolved in 35 ml ethanol and diluted to 50 ml with n-butanol (ACS reagent grade). Ferric chloride, 0.5 gm dissolved in 100 ml concentrated hydrochloric acid. RNA (soluble ribonucleic acid, Schwarz BioResearch) from E. coli. Cadmium chloride, 1.0 and 0.01 M aqueous solutions. Diphenylamine, 1.5 w/v $%; 1.5 gm recrystallized diphenylamine dissolved in 100 ml glacial acetic acid; 1.5 ml concentrated sulfuric acid added, mixed well, and stored cold. 1 Research sponsored by National Institute of General Medical Sciences of National Institutes of Health and the U.S. Atomic Energy Commission under contract with Union Carbide Corporation, Nuclear Division. 42

IMPROVED

METHODS

F’OR RNA

AND DNA

43

Acetaldehyde, 16 mg/ml; 0.5 ml reagent-grade acetaldehyde diluted to 25 ml with water and stored cold. DNA (deoxyribonucleic acid sodium salt, Calbiochem) , highly polymerized, A grade, from salmon. METHODS

Precipitation of Nucleic Acids with Cadmium(H). A sample, from 1 to 10 ml in volume, containing from 20 to 200 pg of nucleic acid per milliliter was transferred to a 15 ml centrifuge tube. Sufficient CdCl, either as a 1 M solution or as a solid, was added to bring the cadmium( II) concentration to 0.2 to 1 M. The final pH was usually between 4 and 9. After standing 5-10 min at 4O”C, it was centrifuged and the supernatant discarded. The precipitate was washed twice with 3 ml of CdCl, (0.1 M) , again discarding the supernatant. Either RNA or DNA was determined by adding the appropriate reagents directly to the precipitate. Occasionally both were determined by first hydrolyzing the precipitate (20 min at 90” in 0.1 N HCl) and using separate portions of the hydrolyzate. Samples containing concentrations of inorganic salts greater than 0.2 M were diluted 2- to 3-fold prior to the cadmium precipitation. Accuracy and precision are not affected significantly by this step. Determination of RNA. 1.0 ml of the orcinol reagent was mixed with 3.0 ml of the ferric chloride reagent in a test tube containing the precipitated nucleic acid (20 to 800 pg) and 1.0 ml of water or containing 1.0 ml of the above hydrolyzate. After mixing thoroughly the tube was put in a 90°C water bath for 60 min. The solution was cooled to room temperature, diluted as necessary, and the absorbance measured against an appropriate reagent blank at 660 nm. Determination of DNA. The chromogenic reagent was prepared by adding 0.10 ml of the acetaldehyde solution to each 20 ml of the diphenylamine solution to be used; 2 ml portions of the freshly prepared chromogenic reagent were added to 20 to 100 pg of precipatated DNA and 1 ml of water or to 1.0 ml aqueous samples and mixed well. The samples were allowed to stand overnight at 3’7°C and the absorbance measured against a reagent blank at 600 nm. RESULTS

Precipitation

of Nucleic

AND

Acids

the efficiency of the precipitation

DISCUSSION

Cadmium(H). We studied procedure as a function of pH,

with

44

HATCHER

AND

GOLDSTEIN

cadmium ion concentration, nucleic acid concentration, and, to a certain extent, the molecular weight of RNA. As shown in Figure 1, the fraction precipitated is the same between pH 2 and 11. Below pH 2 acid precipitation occurs and above pH 10 a bulky precipitate of Cd (OH) 2 appears which makes handling difficult but does not interfere with the nucleic acid precipitation. IOr-

I

I

1

I

I

.

DNA . .

.

0

.

1

.

I

I

I

I

I

2

4

6 PH

8

IO

FIG. 1. Effect of pH on precipitation of tRNA and DNA with cadmium(I1). The percentage unprecipitated was determined by measuring absorbance at 260 nm before and after precipitation. RNA and DNA concentrations were 100 pg/ml and cadmium (II) concentration, 1 M.

The cadmium ion concentration necessary for essentially complete precipitation (Fig. 2) is 0.01 M for DNA and 0.2 M for the RNA’s tested. The molecular weight of the RNA appears to be a factor with the higher molecular weight species more easily precipitated. Mono-, di-, and trinucleotides are not precipitated and the “crude” RNA, which is a mixture of ribosomal and tRNA’s, precipitated at a lower cadmium ion concentration than tRNA alone. We believe that the residual S-10$% of the RNA’s that remains in solution in 1 M cadmium (II) solutions represents oligonucleotide contamination of our samples. Polyacrylamide gel electrophoresis of these samples also indicates the presence of low molecular weight RNA. We should also, point out that, as indicated in Figure 2, when precipitates are washed the wash solution must contain an appropriate concentration of cadmium(I1) or the precipitate iwill redissolve. At low concentrations of nucleic acid, cadmium is a considerably more efficient precipitant than perchloric acid (5)) as shown in Figures 3 and 4. From solutions containing as little as 10 pg of nucleic

IMPROVED

0.001

METHODS

FOR RNA AND

0.01 CADMIUM

3 LO

0.1 (II),

45

DNA

hf

2. Nucleic acid precipitation as function of cadmium(I1) concentration. pH was 7 * 1 and nucleic acid concentration was 100 pg/ml. “Crude” RNA is a mixture of ribosomal and tRNAs. The supernate was analyzed as in Figure 1. FIG.

acid per milliliter, about 90% of the DNA and 80% of the RNA can be precipitated while only about 60% is precipitated by perchloric acid in either case. To confirm these results for DNA, which were based on analysis of the supernate, analysis of the precipitate was carried out as described under “Methods” with the results I

I

I

I

PERCHLORIC

ACID

I

I

I

I

20

40

60

a0

too

FIG. 3. Comparison of cadmium (II) and perchloric acid precipitation of RNA at low concentration. A cadmium(I1) concentration of 0.2 M at pH 7 +- 1, and a perchloric acid concentration of 0.3 M were used. Sample volume was 5 ml. The supernate was analyzed as in Figure 1.

46

HATCHER

AND

25

L

I 0

GOLJXTEIN

1 20

I 40

I 60

DNA,

I 80 .

100

PP

FIG. 4. domparison of cadmium( II) and perchloric DNA at low concentration. Conditions as in Figure 3.

acid precipitation

of

shown in Figure 5. Absorbancies obtained by the diphenylamine method were approximately 50% greater after cadmium (II) precipitation than after perchloric acid precipitation. At nucleic acid concentrations above 200 pg/ml, both precipitation procedures are essentially quantitative. Elimination of Interferences in the Diphenylamine Method. Although many reagents added in the process of separating RNA’s from E. coli cells interfere in the diphenylamine color reaction,

DNA,

5. Analysis of DNA precipitate tation with cadmium (II) or perchloric FIG.

CQ

by diphenylamine method acid from low concentration

after precipisolutions.

IMPROVED

METHODS

FOR RNA

47

AND DNA

DNA can be determined in these extracts following a precipitation step (Table 1). The only difficulty experienced with precipitation resulted from high salt concentrations. Doubling the volume of such samples before precipitating with cadmium eliminated this problem. The insolubility of the cadmium-DNA salt lends itself to this solution. TABLE Removal

of Interfering

1

Substances by Precipitating

Additions M

Sodium acetate Sodium thiosulfate Sodium chloride Glycine Phenol fl-Mercaptoethanol Perchloric acid Ethanol Glutathione n Each solution contained mium-DNA precipitate.

0.2 0.002 0.4 0.4 0.2 0.01 0.2 0.3 0.001 100 pg DNA;

Initially

77 PPt 82 50 28 0 70 30 67

DNA with Cadmium(I1) DNA

found

(/.&a

After

Cd*+ pptn.

After 2:l dilution and Cd*+ pptn.

81 93 38 101 85 90 98 95 87

reagents were added directly

100 87 107 105 90 82 108 84 96 to the cad-

The behavior of cadmium ribonucleates is very nearly the same as cadmium deoxyribonucleates, and cadmium precipitation of RNA can be used to avoid interferences in the orcinol method. One physical difference is that the cadmium-RNA precipitate is redissolved more rapidly than cadmium-DNA by ligands [which compete for cadmium(I1) ions, e.g., ammonia. This difference may have analytical utility. Improvement in the Oricinol Procedure. The three reagents in the conventional orcinol procedure are: (a) concentrated acid, to dehydrate ribose to furfural ; (b) orcinol, to couple with furfural producing a colored product; and (c) ferric ions, as a catalyst. Small changes in the concentrations of these reagents produce significant changes in intensity of the final colored solution (6), particularly changes in the acid concentration (Fig. 6). This dependency of color intensity on acid concentration, however, can be minimized by adding a fourth reagent, n-butanol. In the presence of n-butanol, the color intensity is almost independent of acid concentration between 7.0 and 8.0 N acid (Fig. 6)) and only slightly dependent on n-butanol concentration. Accordingly, our procedure

48

HATCHER

OL

’

6.0

’

6.4

AND GOLDSTEIN

t

’

’

6.6

’

7.2

’

’

7.6

c

’

8.0

HCI, N

6. Effect of HCI concentration on orcinol method for RNA in presence and absence of n-butanol. In one series, butanol was omitted from the orcinol reagent. 200 pg RNA present in each case. FIG.

uses the maximum practical acid concentration of 7.2 N, incorporates the optimum orcinol and ferric chloride concentrations recommended by Miller (6)) and is 1.1 M in n-butanol (added in the orcino1 reagent). We tested the effects of extraneous materials added to E. coli extracts on recovery of RNA (Table 2). The corrected standard deTABLE

2

Effects of Extraneous Materials on Recovery of RNA at Different Acid Concentrations and in the Presence of n-Butanol RNA found (rug)’ Hydrocblorie acid (N) Subgtance

tested

Sodium acetate Sodium chloride Sodium thiosulfateb Magnesium chloride Cadmium chloride EDTA Glutathioneb Isopropanol Phenol* Corrected S.D.c

M

0.2 0.2 0.1 0.2 0.2 0.02 0.01 0.5 0.2

6.0

24.6 26.5 PPt 23.6 19.8 17.5 14.0 37.5 14.3 zt33.1%

7.2

24.4 23.4 PPt 25.7 25.2 25.3 21.9 26.7 14.6 zt4.5270

7.2 + n-butanol

25.3 24.6 PPt 25.6 25.6 24.6 21.5 24.5 19.7 f2.10%

“25.0 mg RNA was used in each test. The recorded values are the average of 3 determinations. *Interference by these materials can be eliminated by the cadmium precipitation procedure. See text for discussion. CValues more than 2.5 times the standard deviation were discarded and the standard deviation was recalculated.

IMPROVED

METHODS

49

FOR RNA AND DNA

viations were 33.1, 4.52, and 2.10%, respectively, for acid concentrations of 6.0 N of 7.2 N , and of 7.2 N plus n-butanol. The presence of butanol, therefore, also stabilized the color intensity. Other alcohols were evaluated but butanol was more soluble than most and a lower concentration was required. Ethanol had no effect. Typical assay results for RNA by this procedure are shown in Table 3. Precision and accuracy seem acceptable with recoveries of standard additions of RNA slightly above 100%. In these and all other samples that contained reducing agents or phenol (see Table 2). cadmium precipitation was used to avoid interference problems. Wildly fluctuating results were obtained for these samples using conventional assay conditions (1). Dilution factors (omitted from the table) varied for each sample as these are fractions taken from a DEAE-cellulose column following a number of preliminary separations. TABLE

3

Typical Analyses of RNA in DEAE-Cellulose

Column Effluents

RNA h&ml) Fraction

Added

Found

1

0 0 40 80 0 0 40 80 0 0 100 100 0 40

65 65 112 142 160 156 202 240 150 150 265 275 255 295

2

3

4

Net

in sample

65 65 72 62 160 156 162 160 150 150 165 175 255 255

It should be mentioned that these methods, in common with other orcinol and diphenylamine methods, measure only the ribose or deoxyribose associated with purine bases. Also, when determining RNA in the presence of DNA, the usual correction must be made to the RNA value based on DNA content. SUMMARY

Nucleic acids (RNA and DNA) can be separated from extraneous materials by precipitation as their cadmium salts, This sepa-

50

HATCHER

AND GOLDSTEIN

ration substantially improves the reliability of subsequent determinations of RNA by the orcinol method and of DNA by the diphenylamine method. In addition, color formation in the orcinol reaction is stabilized by addition of n-butanol to the reaction mixture, thereby improving the precision of the method. ACKNOWLEDGMENT Mr. W. M. Stwartout

provided

able technical

assistance

throughout

this work.

REFERENCES 1. VOLKIN, E., AND COHN, W. E., in “Methods of Biochemical Analysis,” (D. Glick, ed.), Vol. 1, p. 287. Interscience, New York, 1954. 2. BURTON, K., Rio&em. J. 62, 315 (1956). 3. HUTCHINSON, W. C., AND MUNRO, H. N., Analyst 86,768 (1961). 4. MUNRO, H. N., AND FLECK, A., Analyst 91, ‘78 (1966). 5. SHIBKO, S., KOIVISTOINEN, P., TRATNYEK, C. A., NEWHALL, A. R., AND FRIEDMAN, L., Anal. Biochem. 19,514 (1967). 6. MILLER, G. L., GOLDER, R. H., MILLER, E. E., Anal. Chem. 23,903 (1951).