- Email: [email protected]
The fluorometric determination of nucleic acids in pea seeds by use of ethidium bromide complexes
ANALYTICAL
BIOCHEMISTRY
The Fluorometric Pea
Seeds
67. 384-391 (1975)
Determination
by Use of Ethidium
ABDEL-RAHMAN
of Nucleic Bromide
Acids
in
Complexes
A. EL-HAMALAWI, J. STUARTTHOMPSON, AND GEOFFREY R. BARKER
Department of Biochemistry,
The University, Manchester Ml3 9PL, England
Received November 12. 1974; accepted February 18, 1975 A method is described for the determination of RNA and DNA in plant tissues which depends on the effect of complex formation on the Huorescence of ethidium bromide. Previous methods were found to be inapplicable to the analysis of plant material because of the high activity of ribonuclease in tissue extracts. Treatment of extracts with bentonite overcomes this difficulty and also allows measurement of fluorescence without the need to use frontal illumination. The procedure described compares favourably with earlier methods as regards accuracy, sensitivity and simplicity.
Most methods for the determination of nucleic acids in tissues depend on measurement of phosphorus, carbohydrate or extinction due to the bases. All such techniques are subject to interference by materials other than nucleic acids, and the reliability of a variety of published methods has been critically reviewed by Munro and Fleck (1). Of these, the method of Schmidt and Thannhauser (2) is considered to be less subject to error than most but presents difficulties when applied to extracts of plant tissue. The analysis is based on the determination of total precipitable nucleic acid before and after hydrolysis of RNA at alkaline pH: however, Smillie and Krotkov (3) found that there is no correlation between RNA contents of pea tissue based on determinations of pentose, phosphorus and I&, unless contaminating material is removed by ion-exchange chromatography after the alkaline treatment. Barker and Hollinshead (4) found that the ion-exchange treatment could be omitted if corrections were made for the contribution of contaminating protein to measurements of extinction, but neither of these methods involving ionexchange has the simplicity and speed required for studies of changes in the nucIeic acid content to tissues during growth. Moreover, extensive control experiments are necessary before such methods can be applied to tissues of different origins. More recent methods, such as those based on high pressure liquid chromatography of thymine (5) and the reaction of deoxyribose with p-nitrophenylhydrazine (6), do not give the sensitiv384 Copyright All rights
@ 1975 by Academic Press, Inc of reproduction in any form reserved.
FLUOROMETRY
OF
NUCLEIC
ACIDS
385
ity required for studies of nucleic acid metabolism during development and are not applicable to RNA. The method of Sheridan et al. (7) gives high sensitivity but appeared likely to present difficulties if applied to tissue extracts and involves apparatus not commonly available. The most promising methods depend on the enhancement of fluorescence of appropriate dyes by complex formation with nucleic acids. The first such method for the determination of nucleic acids was developed by Le Pecq and Paoletti (8) and depends on the binding of ethidium bromide to nucleic acids. The increase in fluorescence under the conditions of ionic strength and pH used is solely attributable to intercalation (9) and is proportional to the amount of double-stranded nucleic acid present rather than the total nucleic acid. However, this method is applicable only to purified preparations of nucleic acids. Karsten and Wollenberger (10) have described a similar method that can be used for the analysis of extracts of rat liver, but we have found it to be inapplicable to extracts of plant tissue. The present communication describes a modification of Karsten and Wollenberger’s method which has been used successfully with extracts of pea seed. Karsten and Wollenberger used Pronase to remove protein from the tissue extracts before measurement of total nucleic acids. DNA was measured after treatment of the extracts with ribonuclease, and the RNA content was obtained by difference. However, their method for the determination of total nucleic acid is not satisfactory with plant material on account of the high concentration of endogenous ribonuclease. We have found that addition of bentonite to extracts of pea tissue avoids this difficulty, and, after centrifugation, a clear solution is obtained which can be analysed fluorometrically without recourse to the frontal illumination technique which was necessitated in Karsten and Wollenberger’s method by turbidity of the solutions. It is shown that the present modification, when applied to the analysis of plant material, is preferable to the method of Smillie and Krotkov (3). MATERIALS
Ethidium bromide (2,7-diamino-9-phenyllo-ethylphenanthridium bromide), bentonite (technical grade), calf thymus DNA (protein content, < O.l%), yeast RNA and bovine pancreatic ribonuclease (EC 3.1.4.22), protease free, 40-50 Kunitz unitslmg, were purchased from B. D. H. Chemicals Ltd., Poole, U.K. Pronase (Type VI nonspecific protease from Streptomyces gviseus; 3-4 unitslmg) was obtained from Sigma Chemical Co. Ltd., London U.K.). Yeast RNA was purified by the method of Frisch-Niggemeyer and Reddi (11). RNA was prepared from pea seed following the method of Ewing and Cherry ( 12).
386
EL-HAMALAWI,
THOMPSON
AND
BARKER
APPARATUS
Measurements of fluorescence were made with a Model 204 fluorescence spectrometer (Perkin-Elmer Ltd., Beaconsfield, U.K.). In most experiments the sensitivity was set at 8 and the selector at X 10. ANALYTICAL
PROCEDURES
1. Reagents. 1) Saline-acetate buffer. All reactions were carried out in the presence of M sodium acetate buffer, pH 5.6, containing M NaCl. 2) Ethidium bromide, 20 pg/ml in saline-acetate buffer. 3) Pronase, 80 pg/ml in saline-acetate buffer, prepared freshly each day. 4) Ribonuclease, 20 ,ug/ml in saline-acetate buffer. The solution was heated at 80°C for 10 min before use to inactivate any contaminating deoxyribonuclease. 5) Standard DNA, 20 pg/ml in saline-acetate buffer, allowed to dissolve slowly over night at 2-4°C. 6) Bentonite suspension. Bentonite (10 g) was equilibrated with 200 ml of saline-acetate buffer following the method of Brownhill et al. (13). The suspension was vigorously stirred, centrifuged at 9,OOOg and the supernatant fluid discarded. The precipitate was treated in the same way a further four times. The final precipitate was suspended in saline-acetate buffer to give a 5% (w/v) suspension which was stored at -20°C. 2. Preparation of extracts. Cotyledons from five peas were ground in a mortar at 2-4°C with acid-washed sand and 50 ml of saline-acetate buffer. Solids were removed by centrifugation at 2,OOOg for 10 min at 4°C and the supernatant fraction was diluted to 100 ml with saline-acetate buffer. Bentonite suspension (0.5%, w/v; 0.2 ml) was added to 5 ml of the above solution and diluted to 20 ml with saline-acetate buffer. The suspension was shaken at 2-4°C for 30 min and centrifuged at 20,OOOg for 20 min. The supernatant fluid was decanted and used for fluorometric determination of nucleic acids. 3. Reaction mixtures and measurements. Reactions were carried out essentially as described by Karsten and Wollenberger, except that the volumes of solutions were as shown in Table 1. Measurements of fluorescence were made at 580 nm using excitation at 360 nm. The wavelength of maximum emission used for measurement is a reflection of instrumental variables rather than an absolute value and should be checked for the individual instrument employed. Concentrations of nucleic acids were calculated following Karsten and Wollenberger (lo), using the equations: astd(fE &VA
-fB
-fF
fA-fB
a
+fc)
[II
=
astd(fD
-
’ fd
RNA= 0.46cfA -fB)’
FLUOROMETRY
OF NUCLEIC TABLE
387
ACIDS
1
COMPOSITION OF SOLUTIONS FORMEASUREMENTS OFFLUORESCENCE Final concentration (Pdml)
Solution
Component
Volume (ml)
A
Standard DNA Pronase Buffer Ethidium bromide
1 1 1 1
5 20 5
B
Pronase Buffer Ethidium bromide
I 2 1
20 5
c
Buffer
4
D
Homogenate Pronase Buffer Ethidium bromide
1 1 1 1
20 5
E
Homogenate Pronase Ribonuclease Ethidium bromide
1 1 1 1
20 5 5
F
Homogenate Buffer
1 3
-
where aDNA= amount of DNA per mixture E (pg), astd = amount of standard DNA per mixture A (pg), f= fluorescence intensity (units). In agreement with Karsten and Wollenberger, we find that the factor 0.46 takes into account the use of DNA as standard in the determination of RNA. RESULTS 1. Recovery of DNA and RNA Added to Homogenates and Linearity of Response
Varying quantities of DNA or RNA were added to homogenates from five pairs of pea cotyledons prior to the addition of bentonite as described above. Measurements of fluorescence were made and recovery of nucleic acids calculated. Figure 1 shows that recovery of added nucleic acid is complete. In the experiments described, 0.05-0.2 ml of 5% (w/v) bentonite suspension was found to be optimal for removal of nucleases from the five pairs of cotyledons used and for reducing background fluorescence to an
388
EL-HAMALAWI.
THOMPSON
0 I pg
2 3 4 5 6 Nuclerc acid added/ml
AND
BARKER
7 8 9 10 homogenate
FIG. 1. Recoveries of DNA and RNA added to homogenates of pea cotyledons. Varying quantities of DNA or RNA were added to homogenates of pea cotyledons and DNA (0) and RNA (m) were determined in samples of the homogenates by the fluorometric method described in the text.
acceptable level; if sufficient bentonite was used to reduce the background to zero, some adsorption of nucleic acids occurred. Varying volumes of solution obtained after treatment of homogenates with bentonite were diluted to 1 ml with saline-acetate buffer and used for fluorometric measurement of DNA and RNA. Figure 2 shows a linear correlation between the volume of homogenate used and the amounts of DNA and RNA calculated from measurements of fluorescence. 2. Comparison of the Fluorometric Method with Smillie and Krotkov’s Method Various volumes of eluates from the ion-exchange column and various samples of perchloric acid-hydrolysed precipitate B covering the range
00
02 Volume
04 0.6 of homogenafe
0.8 [ml)
IO
FIG. 2. Correlation of volume of homogenate from pea cotyledons analysed and concentration of DNA (0) and RNA (I) found by the fluorometric method described in the text.
14 k-
FLUOROMETRY
4 E cc 0. E
OF
NUCLEIC
389
ACIDS
- 140
I2
- 120
10
- 100
08
-80
06
.60
0
2 0 Y
04
- 40
02
- 20 0
0
02
04
Volume
0.6
of
08
homogenate
IO
0
imll
FIG. 3. Correlation of volume of homogenate from pea cotyledons analysed and concentration of DNA (0) and RNA (m) found by the method of Smillie and Krotkov.
5-100 ~g of DNA, both prepared as described by Smillie and Krotkov (3), were used to test the linearity of response in their method. The results shown in Fig. 3 indicate that the correlation is not linear at low levels of DNA or RNA. The sensitivities and accuracies of the two methods were also compared as follows. Four homogenates were prepared, and three samples were taken in duplicate from each, within the appropriate ranges of volume, and analysed for RNA and DNA by the two methods. Table 2 shows the standard deviations for each set of analyses. Finally, cotyledons from peas taken at different stages of germination were analysed by the two methods, and the results obtained with samples taken in triplicate are shown in Table 3. DISCUSSION
Figures 1 and 2 show that the fluorometric method described gives a linear correlation with DNA and RNA added to homogenates and with the volume of homogenate used in the determination. By comparison, the method of Smillie and Krotkov does not give a linear correlation for quantities of DNA below 25 pg and of RNA below 400 pug (Fig. 3). The results shown in Table 2 indicate that, by use of the fluorometric method, reproducibility is acceptable down to approximately 2 p.g of RNA and 0.5 pg of DNA per ml of solution analysed. In contrast, the TABLE VARIATION
OF STANDARD ACIDS
DEVIATION IN
THE
2 WITH
CONCENTRATION
SOLUTIONS
Fluorometric
OF
NUCLEIC
ANALYSED
method
Smillie-Krotkov
method
Concentration of RNA (pg/ml) Standard deviation (%)
12.8 0.1
7.7 0.2
2.0 0.8
0.2 28
1310 1.2
869 5.4
530 33
Concentration of DNA (pg/ml) Standard deviation (‘Z)
3.2 0.6
1.9 1.1
0.5 1.5
0.04 30
93 5.4
43 8.9
17 40
452 42 -
390
EL-HAMALAWI,
DETERMINATION
OF
DIFFERENT
THOMPSON
RNA
TABLE 3 AND DNA
STAGES
DURING
AND
BARKER
IN PEA
COTYLEDONS
AT
GERMJNATJON
MG of nucleic acid/cotyledon pair” Age of germinating pea cotyledons (days)
Method of analysis”
0
F SK
Dry
peas 2
7
12
RNA
DNA
1.32 2 0.002
0.25 k 0.003 0.24 2 0.02
1.29 k 0.07
F SK
0.88 2 0.002
0.91 f 0.05
0.23 + 0.002 0.22 2 0.02
F
SK
0.44 " 0.001 0.46 '- 0.03
0.18 2 0.002 0.21 5 0.04
F SK
0.16 ? 0.001 0.24
0.03 " 0.0004 0.03 k 0.005
2 0.08
B Fluorometric method, F; Smillie-Krotkov method, SK. b Results are the means of three determinations with the standard deviation shown.
method of Smillie and Krotkov is unsatisfactory below concentrations of approximately 0.9 mg of RNA/ml and 0.8 mg of DNA/ml. As shown in Table 3, the two methods give comparable results in the analysis of cotyledon tissue if used within their respective ranges. The present method is therefore preferable as regards both sensitivity and reproducibility as well as simplicity in the manipulations. The method can also be used for homogenates containing less RNA and DNA per ml without loss of accuracy by diluting the solutions of ribonuclease, Pronase and ethidium bromide fivefold, increasing the setting of the sensitivity control to 11 and using a standard solution of DNA containing 4 pg/ml for setting the reading of the galvanometer to 100. In conclusion, the method described by Karsten and Wollenberger (IO) for the determination of RNA and DNA in animal tissues has been adapted for use successfully with pea cotyledons. It gives results in close agreement with the less sensitive and more tedious method of Smillie and Krotkov when applied to dry peas or seedlings up to 12 days old. It has the advantage compared with Karsten and Wollenberger’s method that it does not necessitate the use of frontal illumination in the measurement of fluorescence. ACKNOWLEDGMENTS Grants to G. R. Barker and J. S. Thompson from the Science Research Council are gratefully acknowledged. A.-R. A. El-Hamalawi thanks the University of Manchester for the award of a research studentship.
FLUOROMETRY
OF NUCLEIC
ACIDS
391
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Munro, H. N., and Fleck, A. (1966) Analyst 91, 78-91. Schmidt, G., and Thannhauser, S. J. (1945) J. Biol. Chem. 161, 83-89. Smillie, R. K., and Krotkov, G. (I 960) Can. J. But. 38, 3 l-49. Barker, G. R., and Hollinshead, J. A. (1964) Biochem. J. 93, 78-83. Gauche]. G., Gauche], F. D., Beyermann, K., and Kahn, R. K. (1972) Z. Anul. Chem. 259, 183-I 87. Martin, R. F., Donohue, D. C., and Finch, L. R. (1972)AnaI. Biochem. 47, 562-574. Sheridan, R. E., O’Donnell, C. M., and Pautler. E. L. (1973) Anal. Biochem 52, 657-659. Le Pecq. J.-B., and Paoletti, C. (1966) Anal. Biochem. 17, 100-107. Le Pecq, J.-B., and Paoletti, C. (1967)5. Mol. Biol. 27, 87-106. Karsten, U.. and Wollenberger, A. (1972) Anal. Biochem. 46, 135-148. Frisch-Niggemeyer, W. and Reddi, K. K. (1957) B&him. Biophys. Acta 26, 40-46. Ewing, E., and Cherry, J. H. (1968) Biochim. Biophys. Acta 161, 331-340. Brownhill. T. J., Jones. A. S., and Stacey, M. (1959) Biochem. J. 73, 434-438.
BIOCHEMISTRY
The Fluorometric Pea
Seeds
67. 384-391 (1975)
Determination
by Use of Ethidium
ABDEL-RAHMAN
of Nucleic Bromide
Acids
in
Complexes
A. EL-HAMALAWI, J. STUARTTHOMPSON, AND GEOFFREY R. BARKER
Department of Biochemistry,
The University, Manchester Ml3 9PL, England
Received November 12. 1974; accepted February 18, 1975 A method is described for the determination of RNA and DNA in plant tissues which depends on the effect of complex formation on the Huorescence of ethidium bromide. Previous methods were found to be inapplicable to the analysis of plant material because of the high activity of ribonuclease in tissue extracts. Treatment of extracts with bentonite overcomes this difficulty and also allows measurement of fluorescence without the need to use frontal illumination. The procedure described compares favourably with earlier methods as regards accuracy, sensitivity and simplicity.
Most methods for the determination of nucleic acids in tissues depend on measurement of phosphorus, carbohydrate or extinction due to the bases. All such techniques are subject to interference by materials other than nucleic acids, and the reliability of a variety of published methods has been critically reviewed by Munro and Fleck (1). Of these, the method of Schmidt and Thannhauser (2) is considered to be less subject to error than most but presents difficulties when applied to extracts of plant tissue. The analysis is based on the determination of total precipitable nucleic acid before and after hydrolysis of RNA at alkaline pH: however, Smillie and Krotkov (3) found that there is no correlation between RNA contents of pea tissue based on determinations of pentose, phosphorus and I&, unless contaminating material is removed by ion-exchange chromatography after the alkaline treatment. Barker and Hollinshead (4) found that the ion-exchange treatment could be omitted if corrections were made for the contribution of contaminating protein to measurements of extinction, but neither of these methods involving ionexchange has the simplicity and speed required for studies of changes in the nucIeic acid content to tissues during growth. Moreover, extensive control experiments are necessary before such methods can be applied to tissues of different origins. More recent methods, such as those based on high pressure liquid chromatography of thymine (5) and the reaction of deoxyribose with p-nitrophenylhydrazine (6), do not give the sensitiv384 Copyright All rights
@ 1975 by Academic Press, Inc of reproduction in any form reserved.
FLUOROMETRY
OF
NUCLEIC
ACIDS
385
ity required for studies of nucleic acid metabolism during development and are not applicable to RNA. The method of Sheridan et al. (7) gives high sensitivity but appeared likely to present difficulties if applied to tissue extracts and involves apparatus not commonly available. The most promising methods depend on the enhancement of fluorescence of appropriate dyes by complex formation with nucleic acids. The first such method for the determination of nucleic acids was developed by Le Pecq and Paoletti (8) and depends on the binding of ethidium bromide to nucleic acids. The increase in fluorescence under the conditions of ionic strength and pH used is solely attributable to intercalation (9) and is proportional to the amount of double-stranded nucleic acid present rather than the total nucleic acid. However, this method is applicable only to purified preparations of nucleic acids. Karsten and Wollenberger (10) have described a similar method that can be used for the analysis of extracts of rat liver, but we have found it to be inapplicable to extracts of plant tissue. The present communication describes a modification of Karsten and Wollenberger’s method which has been used successfully with extracts of pea seed. Karsten and Wollenberger used Pronase to remove protein from the tissue extracts before measurement of total nucleic acids. DNA was measured after treatment of the extracts with ribonuclease, and the RNA content was obtained by difference. However, their method for the determination of total nucleic acid is not satisfactory with plant material on account of the high concentration of endogenous ribonuclease. We have found that addition of bentonite to extracts of pea tissue avoids this difficulty, and, after centrifugation, a clear solution is obtained which can be analysed fluorometrically without recourse to the frontal illumination technique which was necessitated in Karsten and Wollenberger’s method by turbidity of the solutions. It is shown that the present modification, when applied to the analysis of plant material, is preferable to the method of Smillie and Krotkov (3). MATERIALS
Ethidium bromide (2,7-diamino-9-phenyllo-ethylphenanthridium bromide), bentonite (technical grade), calf thymus DNA (protein content, < O.l%), yeast RNA and bovine pancreatic ribonuclease (EC 3.1.4.22), protease free, 40-50 Kunitz unitslmg, were purchased from B. D. H. Chemicals Ltd., Poole, U.K. Pronase (Type VI nonspecific protease from Streptomyces gviseus; 3-4 unitslmg) was obtained from Sigma Chemical Co. Ltd., London U.K.). Yeast RNA was purified by the method of Frisch-Niggemeyer and Reddi (11). RNA was prepared from pea seed following the method of Ewing and Cherry ( 12).
386
EL-HAMALAWI,
THOMPSON
AND
BARKER
APPARATUS
Measurements of fluorescence were made with a Model 204 fluorescence spectrometer (Perkin-Elmer Ltd., Beaconsfield, U.K.). In most experiments the sensitivity was set at 8 and the selector at X 10. ANALYTICAL
PROCEDURES
1. Reagents. 1) Saline-acetate buffer. All reactions were carried out in the presence of M sodium acetate buffer, pH 5.6, containing M NaCl. 2) Ethidium bromide, 20 pg/ml in saline-acetate buffer. 3) Pronase, 80 pg/ml in saline-acetate buffer, prepared freshly each day. 4) Ribonuclease, 20 ,ug/ml in saline-acetate buffer. The solution was heated at 80°C for 10 min before use to inactivate any contaminating deoxyribonuclease. 5) Standard DNA, 20 pg/ml in saline-acetate buffer, allowed to dissolve slowly over night at 2-4°C. 6) Bentonite suspension. Bentonite (10 g) was equilibrated with 200 ml of saline-acetate buffer following the method of Brownhill et al. (13). The suspension was vigorously stirred, centrifuged at 9,OOOg and the supernatant fluid discarded. The precipitate was treated in the same way a further four times. The final precipitate was suspended in saline-acetate buffer to give a 5% (w/v) suspension which was stored at -20°C. 2. Preparation of extracts. Cotyledons from five peas were ground in a mortar at 2-4°C with acid-washed sand and 50 ml of saline-acetate buffer. Solids were removed by centrifugation at 2,OOOg for 10 min at 4°C and the supernatant fraction was diluted to 100 ml with saline-acetate buffer. Bentonite suspension (0.5%, w/v; 0.2 ml) was added to 5 ml of the above solution and diluted to 20 ml with saline-acetate buffer. The suspension was shaken at 2-4°C for 30 min and centrifuged at 20,OOOg for 20 min. The supernatant fluid was decanted and used for fluorometric determination of nucleic acids. 3. Reaction mixtures and measurements. Reactions were carried out essentially as described by Karsten and Wollenberger, except that the volumes of solutions were as shown in Table 1. Measurements of fluorescence were made at 580 nm using excitation at 360 nm. The wavelength of maximum emission used for measurement is a reflection of instrumental variables rather than an absolute value and should be checked for the individual instrument employed. Concentrations of nucleic acids were calculated following Karsten and Wollenberger (lo), using the equations: astd(fE &VA
-fB
-fF
fA-fB
a
+fc)
[II
=
astd(fD
-
’ fd
RNA= 0.46cfA -fB)’
FLUOROMETRY
OF NUCLEIC TABLE
387
ACIDS
1
COMPOSITION OF SOLUTIONS FORMEASUREMENTS OFFLUORESCENCE Final concentration (Pdml)
Solution
Component
Volume (ml)
A
Standard DNA Pronase Buffer Ethidium bromide
1 1 1 1
5 20 5
B
Pronase Buffer Ethidium bromide
I 2 1
20 5
c
Buffer
4
D
Homogenate Pronase Buffer Ethidium bromide
1 1 1 1
20 5
E
Homogenate Pronase Ribonuclease Ethidium bromide
1 1 1 1
20 5 5
F
Homogenate Buffer
1 3
-
where aDNA= amount of DNA per mixture E (pg), astd = amount of standard DNA per mixture A (pg), f= fluorescence intensity (units). In agreement with Karsten and Wollenberger, we find that the factor 0.46 takes into account the use of DNA as standard in the determination of RNA. RESULTS 1. Recovery of DNA and RNA Added to Homogenates and Linearity of Response
Varying quantities of DNA or RNA were added to homogenates from five pairs of pea cotyledons prior to the addition of bentonite as described above. Measurements of fluorescence were made and recovery of nucleic acids calculated. Figure 1 shows that recovery of added nucleic acid is complete. In the experiments described, 0.05-0.2 ml of 5% (w/v) bentonite suspension was found to be optimal for removal of nucleases from the five pairs of cotyledons used and for reducing background fluorescence to an
388
EL-HAMALAWI.
THOMPSON
0 I pg
2 3 4 5 6 Nuclerc acid added/ml
AND
BARKER
7 8 9 10 homogenate
FIG. 1. Recoveries of DNA and RNA added to homogenates of pea cotyledons. Varying quantities of DNA or RNA were added to homogenates of pea cotyledons and DNA (0) and RNA (m) were determined in samples of the homogenates by the fluorometric method described in the text.
acceptable level; if sufficient bentonite was used to reduce the background to zero, some adsorption of nucleic acids occurred. Varying volumes of solution obtained after treatment of homogenates with bentonite were diluted to 1 ml with saline-acetate buffer and used for fluorometric measurement of DNA and RNA. Figure 2 shows a linear correlation between the volume of homogenate used and the amounts of DNA and RNA calculated from measurements of fluorescence. 2. Comparison of the Fluorometric Method with Smillie and Krotkov’s Method Various volumes of eluates from the ion-exchange column and various samples of perchloric acid-hydrolysed precipitate B covering the range
00
02 Volume
04 0.6 of homogenafe
0.8 [ml)
IO
FIG. 2. Correlation of volume of homogenate from pea cotyledons analysed and concentration of DNA (0) and RNA (I) found by the fluorometric method described in the text.
14 k-
FLUOROMETRY
4 E cc 0. E
OF
NUCLEIC
389
ACIDS
- 140
I2
- 120
10
- 100
08
-80
06
.60
0
2 0 Y
04
- 40
02
- 20 0
0
02
04
Volume
0.6
of
08
homogenate
IO
0
imll
FIG. 3. Correlation of volume of homogenate from pea cotyledons analysed and concentration of DNA (0) and RNA (m) found by the method of Smillie and Krotkov.
5-100 ~g of DNA, both prepared as described by Smillie and Krotkov (3), were used to test the linearity of response in their method. The results shown in Fig. 3 indicate that the correlation is not linear at low levels of DNA or RNA. The sensitivities and accuracies of the two methods were also compared as follows. Four homogenates were prepared, and three samples were taken in duplicate from each, within the appropriate ranges of volume, and analysed for RNA and DNA by the two methods. Table 2 shows the standard deviations for each set of analyses. Finally, cotyledons from peas taken at different stages of germination were analysed by the two methods, and the results obtained with samples taken in triplicate are shown in Table 3. DISCUSSION
Figures 1 and 2 show that the fluorometric method described gives a linear correlation with DNA and RNA added to homogenates and with the volume of homogenate used in the determination. By comparison, the method of Smillie and Krotkov does not give a linear correlation for quantities of DNA below 25 pg and of RNA below 400 pug (Fig. 3). The results shown in Table 2 indicate that, by use of the fluorometric method, reproducibility is acceptable down to approximately 2 p.g of RNA and 0.5 pg of DNA per ml of solution analysed. In contrast, the TABLE VARIATION
OF STANDARD ACIDS
DEVIATION IN
THE
2 WITH
CONCENTRATION
SOLUTIONS
Fluorometric
OF
NUCLEIC
ANALYSED
method
Smillie-Krotkov
method
Concentration of RNA (pg/ml) Standard deviation (%)
12.8 0.1
7.7 0.2
2.0 0.8
0.2 28
1310 1.2
869 5.4
530 33
Concentration of DNA (pg/ml) Standard deviation (‘Z)
3.2 0.6
1.9 1.1
0.5 1.5
0.04 30
93 5.4
43 8.9
17 40
452 42 -
390
EL-HAMALAWI,
DETERMINATION
OF
DIFFERENT
THOMPSON
RNA
TABLE 3 AND DNA
STAGES
DURING
AND
BARKER
IN PEA
COTYLEDONS
AT
GERMJNATJON
MG of nucleic acid/cotyledon pair” Age of germinating pea cotyledons (days)
Method of analysis”
0
F SK
Dry
peas 2
7
12
RNA
DNA
1.32 2 0.002
0.25 k 0.003 0.24 2 0.02
1.29 k 0.07
F SK
0.88 2 0.002
0.91 f 0.05
0.23 + 0.002 0.22 2 0.02
F
SK
0.44 " 0.001 0.46 '- 0.03
0.18 2 0.002 0.21 5 0.04
F SK
0.16 ? 0.001 0.24
0.03 " 0.0004 0.03 k 0.005
2 0.08
B Fluorometric method, F; Smillie-Krotkov method, SK. b Results are the means of three determinations with the standard deviation shown.
method of Smillie and Krotkov is unsatisfactory below concentrations of approximately 0.9 mg of RNA/ml and 0.8 mg of DNA/ml. As shown in Table 3, the two methods give comparable results in the analysis of cotyledon tissue if used within their respective ranges. The present method is therefore preferable as regards both sensitivity and reproducibility as well as simplicity in the manipulations. The method can also be used for homogenates containing less RNA and DNA per ml without loss of accuracy by diluting the solutions of ribonuclease, Pronase and ethidium bromide fivefold, increasing the setting of the sensitivity control to 11 and using a standard solution of DNA containing 4 pg/ml for setting the reading of the galvanometer to 100. In conclusion, the method described by Karsten and Wollenberger (IO) for the determination of RNA and DNA in animal tissues has been adapted for use successfully with pea cotyledons. It gives results in close agreement with the less sensitive and more tedious method of Smillie and Krotkov when applied to dry peas or seedlings up to 12 days old. It has the advantage compared with Karsten and Wollenberger’s method that it does not necessitate the use of frontal illumination in the measurement of fluorescence. ACKNOWLEDGMENTS Grants to G. R. Barker and J. S. Thompson from the Science Research Council are gratefully acknowledged. A.-R. A. El-Hamalawi thanks the University of Manchester for the award of a research studentship.
FLUOROMETRY
OF NUCLEIC
ACIDS
391
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Munro, H. N., and Fleck, A. (1966) Analyst 91, 78-91. Schmidt, G., and Thannhauser, S. J. (1945) J. Biol. Chem. 161, 83-89. Smillie, R. K., and Krotkov, G. (I 960) Can. J. But. 38, 3 l-49. Barker, G. R., and Hollinshead, J. A. (1964) Biochem. J. 93, 78-83. Gauche]. G., Gauche], F. D., Beyermann, K., and Kahn, R. K. (1972) Z. Anul. Chem. 259, 183-I 87. Martin, R. F., Donohue, D. C., and Finch, L. R. (1972)AnaI. Biochem. 47, 562-574. Sheridan, R. E., O’Donnell, C. M., and Pautler. E. L. (1973) Anal. Biochem 52, 657-659. Le Pecq. J.-B., and Paoletti, C. (1966) Anal. Biochem. 17, 100-107. Le Pecq, J.-B., and Paoletti, C. (1967)5. Mol. Biol. 27, 87-106. Karsten, U.. and Wollenberger, A. (1972) Anal. Biochem. 46, 135-148. Frisch-Niggemeyer, W. and Reddi, K. K. (1957) B&him. Biophys. Acta 26, 40-46. Ewing, E., and Cherry, J. H. (1968) Biochim. Biophys. Acta 161, 331-340. Brownhill. T. J., Jones. A. S., and Stacey, M. (1959) Biochem. J. 73, 434-438.