ANALYTICAL

BIOCHEMISTRY

199,

137-141

(1991)

Quantitation of DNA and RNA in Crude Tissue Extracts by Flow Injection Analysis Elaine

M. Caldarone

and Lawrence

J. Buckley

National Oceanic and Atmospheric Administration, National Marine Narragansett Laboratory, Narragansett, Rhode Island 02882-l 199

Received

May

Service, Northeast

Fisheries

Center,

14, 1991

An automated two-dye flow injection analysis system to quantitate DNA and RNA in crude extracts of tissues is described. The method uses the fluorochrome dyes ethidium bromide and Hoechst 33258. DNA concentration is determined directly from its fluorescence in Hoechst dye. RNA is estimated from fluorescence in ethidium bromide after subtraction of the fluorescence due to DNA. This method has several advantages: a simple extraction procedure, a low detection limit (0.01 pg DNA and 0.10 pg RNA), automation, and a high sample Q 1991 Academic Press. Inc. throughput.

Several methods for determining the concentration of nucleic acids in organisms have been developed. Methods based on the uv absorption of nucleic acids are limited by sample size (l), while more sensitive fluorometric assays typically require an extensive amount of operator attention or a lengthy extraction procedure (2,3). Murray and Paaren (4) described a flow injection analysis (FIA)l system with fluorometric detection for the analysis of purified nucleic acids. In this paper we describe an optimized FIA system for the estimation of DNA and RNA levels in crude extracts of biological tislsues. Our method uses a simple N-lauroylsarcosine extraction procedure and automated sample and data handling. The fluorophors ethidium bromide (EB) and Hoechst 33258 (Hoechst) are used for detecting the nucleic acids. EB is an intercalating agent that binds specifically to double-stranded regions of polynucleotides (5,6). Hoechst preferentially binds to adenine-thymine base areas, which occur only in DNA (7,8).

1 Abbreviations used: FIA, bromide; WF, winter flounder; 0003-2697/91 Copyright All rights

Fisheries

flow injection analysis; CT, calf thymus; CL,

$3.00 8 1991 by Academic Press, of reproduction in any form

EB, ethidium calf liver.

Application to DNA and RNA quantitation in larval and juvenile fish is presented and limitations of this system are discussed. EXPERIMENTAL

PROCEDURE

Materials. Materials were obtained from the following sources: N-lauroylsarcosine sodium salt, Trizma base [Tris(hydroxymethyl)aminomethane], RNA (Type IV, calf liver), RNA (Baker’s yeast), DNA (Type III, Na salt, salmon testes), EDTA (free acid) from Sigma’; ethidium bromide (2,7-diamino-lo-ethyl-g-phenylphenanthridium bromide), RNA (phage MS 2, DNA free), RNase (ribonuclease, DNase free), DNA (high molecular weight, calf thymus), and DNase I (RNase free) from Boehringer-Mannheim; and Hoechst Dye H33258 (2[2-(4-hydroxyphenyl)-6-benzimidazoyl]-6-(l-methyl-4piperazyl)-benzimidazole, trihydrochloride) from Calbiochem. All other common chemicals such as NaCl and HCl were analytical grade. All solutions were prepared in glass-distilled, deionized water using glassware that had previously been heated to 250°C for 4 h. Preparation of reagents and standards. The Hoechst reagent containing 5 mM Tris-HCl, 0.5 mM EDTA, pH 7.5 (Tris-EDTA buffer), 0.2 N NaCl, and 50 pg/liter Hoechst was prepared daily. The EB reagent containing Tris-EDTA buffer and 137.5 pg/liter EB was stable for several days when stored at 4’C. Working standards of RNA and DNA were prepared daily by serially diluting previously frozen stock solutions with 0.1% sarcosine (N-lauroylsarcosine) in TrisEDTA buffer. DNA standards ranged from 0.36 to 2.67 pg/ml; RNA standards ranged from 3.65 to 27.37 pg/ml. Concentrations of the stocks were determined spectro-

‘Reference United States

to trade names Government.

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138

FIG.

CALDARONE

1.

Schematic

diagram

of the flow injection

analysis

system.

photometrically at 260 nm using a value of E::m = 200 for high-molecular-weight RNA and DNA (9). Flow injection system. A schematic diagram of the flow injection system is shown in Fig. 1. The fluorochrome reagent was pumped by a medium-pressure piston pump (LDC/Milton Roy) at a rate of 1.5 ml/min to an autoinjection valve (Valco VICI) equipped with a 50~1 sample loop. An autosampler (ISCO, ISIS) and peristaltic pump delivered the sample to the valve and washed the loop with water between injections. The autosampler was also used to time the injection valve and peristaltic pump. The sample mixed with the fluorochrome reagent while being pumped through Teflon microbore tubing (180 cm of 0.5-mm-i.d. tubing) to the fluorescence detector (Perkin-Elmer 650-10s equipped with an 18-~1 flow cell). For the EB reagent, the excitation wavelength was set at 525 nm, the emission at 600 nm, and a 20-nm slit width and a range of 1.0 were used. An excitation wavelength of 356 nm, an emission of 458 nm, a 15-nm slit width, and a range of 0.3 were used with the Hoechst reagent. To prevent any carryover of dye during switching between reagents, the FIA system was flushed with 20 ml of water, followed by 30 ml of 0.26% sodium hypochlorite, and another 20 ml of water. Data collection and calculations. The output from the fluorometer was connected to a PC equipped with Labtech Notebook data acquisition and Labtech Chrom chromatography integrator and data archive software packages (Laboratory Technologies Corp.). Using the peak areas calculated by these programs, the sample DNA concentration was determined directly from a DNA-Hoechst calibration curve. The contribution of DNA-EB fluorescence to the total fluorescence in EB was estimated from the DNA concentration determined above and the DNA-EB calibration curve. Subtracting the estimated DNA-EB fluorescence from the corre-

AND

BUCKLEY

sponding total-EB fluorescence gives the RNA-EB fluorescence. RNA values were determined using an RNAEB calibration curve. Calf liver RNA and calf thymus DNA were used as standards. The calculations were automated using macros in a Lotus l-2-3 spreadsheet (Lotus Development Corp.). Animals. Tissues used in the optimization of this system were sand lance (Ammodytes americanus) liver and whole larvae and winter flounder (Pseudopleuronectes americanus) liver, muscle, eggs, and whole larvae. Larval fish were extracted in 0.15 to 0.45 ml of 1% sarcosine-Tris-EDTA buffer (depending upon size) and diluted with a volume of Tris-EDTA buffer to give a final sarcosine concentration of 0.1%. After centrifugation the supernatant was placed in the autosampler (Fig. 2). Liver, muscle, and eggs were diluted 1:lO with ice-cold distilled water and homogenized in an SDT Tissumizer with three 15-s pulses at maximum power. SarcosineTris-EDTA was added to aliquots of the homogenates to yield a 1% sarcosine concentration. The solutions were incubated at room temperature as outlined in Fig. 2 and diluted with Tris-EDTA buffer to yield a final concentration of 0.1% sarcosine and a tissue concentration of approximately 0.8,10.0, and 0.10 mg/ml, respectively.

RESULTS

Optimization of reagent and buffer. The concentrations of Hoechst (50 pg/liter) and EB (137.5 vg/liter) were chosen to minimize the background and 0.1% sarcosine blank fluorescence while maintaining a liner response to the standards and a high sample fluorescence. The optimum pH for the extraction and reagent buffer was selected experimentally from six values for Hoechst and four values for EB. A high fluorescence yield and a stable sample-dye complex occurred at a pH of 7.5. NaCl (0.2 N) was added to the Hoechst reagent to enhance the stability of the DNA-dye complex and suppress the fluorescence of RNA (10). NaCl was not added

by&kiyk Egd 1 $$$&g@ 1 VubrtEi 1 1 4 1 1 I k YolithYT FIG.

2.

Flow

chart

I5 min at mom telrperature Mrtex vigomusly 15 min at mom terrperature 1.35ml Tris-EDTAhuller Shake Centriluge 5 min at 2,500xg SUPEdATANT to autosampler

of the IV-laurolysarcosine

extraction

procedure.

FLOW

INJECTION

ANALYSIS

to the EB reagent because the blank fluorescence rose above acceptable levels. Emission scans of eggs, larvae, fish tissues, and RNA and DNA standards in the presence of EB showed maximum fluorescence at 580-600 nm. Maximum fluorescence occurred at an excitation wavelength of 525 nm. In the presence of Hoechst, emission and excitation maxima were at 435-458 and 343-356 nm, respectively. Sarcosine conOptimization of sarcosine extraction. centrations greater then 0.1% increased the blank fluorescence above acceptable levels in both of the dyes; however, significantly more nucleic acids were extracted at concentrations of 1 and 2%. For this reason, samples were extracted in 1.0% N-lauroylsarcosineTris-EDTA buffer and then diluted IO-fold with TrisEDTA buffer before FIA. Incubation times longer than 30 min did not affect the extraction efficiency of larval fish up to 7 mm total length (0.2 mg dry weight). Nucleic acids were extracted from larger fish more effectively by either doubling the incubation time to 60 min or homogenizing and treating the fish the same as muscle samples. Recoveries of RNA and DNA from a second extraction of the pellet were less than 1.0% of the values obtained from the first extraction. RecovRecovery of spikes, sensitivity, and precision. ery of added calf thymus DNA from larval samples was 97.9 k 1.3% in Hoechst and 101.5 +- 1.4% in EB. Recovery of calf liver RNA was 98 f 2.9% in EB. Fluorescence in EB of a mixture of calf liver RNA and calf thymus DNA was 99 & 0.8% of the sum of their individual fluorescence. The fluorescence in EB and Hoechst of a pooled extract increased in proportion to the amount of tissue extract analyzed.

FIG. 3. Observed signal from the fluorometer after injection of nucleic acid standards. (A) Calibration curve for calf thymus DNA in Hoechst (2.7-0.7 pg/ml); (B) calibration curve for calf liver RNA in EB (27.4-3.6 pglml); (C) calibration curve for calf thymus DNA in EB (2.7-0.3 pg/ml).

OF

DNA

AND

139

RNA TABLE

1

Average Slope and Intercept of Standard Curves Generated in EB by Commercially Available Nucleic Acids Average slope

Average intercept

Calf liver RNA Baker’s yeast RNA MS 2 RNA

0.361 0.569 1.023

0.05 0.21 0.35

Salmon sperm DNA Calf thymus DNA

0.777 1.879

0.07 -0.06

With automated injection, the RNA and DNA content of individual larvae as small as 20 pug dry weight could be determined (approximately 0.9 pg RNA, 0.2 pg DNA total content). Hand-injected samples require several-fold less tissue. The average coefficient of variation (sample standard deviation as a percentage of the mean) of 10 subsamples of a pooled homogenate was 3.2% for DNA and 2.9% for RNA within 1 day and 3.6 and 5.0%, respectively, when the samples were run on 6 separate days over a period of 3 weeks. Comparison of standards. All RNA and DNA standards tested produced linear calibration curves with the fluorochrome reagents (Fig. 3); however, the slopes and intercepts were different (Table 1). Contamination of the calf liver RNA with DNA was determined by analysis in Hoechst. Contamination averaged ~1.0% of the spectrophotometrically determined value, when compared to the calf thymus DNA standard. The addition of DNase to the calf liver RNA eliminated all fluorescence in Hoechst. Residual fluorescence after treatment with RNase and DNase. Several sets of tissue samples and standards were treated with DNase and RNase to determine the residual fluorescence using a modification of Bentle et al. (2). The nucleic acid-fluorophor fluorescence of the standards and larval fish extracts were virtually eliminated in both dyes (Table 2). When the enzymes were added to most tissues, the fluorescence remaining was equal to the endogenous fluorescence (described below) and increased in proportion to the sample concentration. Endogenous sample fluorescence. Samples of larvae, fish tissues, and standards were analyzed at the two excitation and emission settings, using buffers with and without the fluorescent dyes. The endogenous fluorescence (fluorescence without dye) of the tissues and standards as a percentage of the nucleic acid-fluorophor fluorescence value is shown in Table 2. The endogenous fluorescence of the different tissue extracts, at both the Hoechst and the EB excitation and emission settings, increased in proportion to the sample concen-

140

CALDARONE TABLE

2

The Endogenous and Residual Fluorescence of Standards and Tissues as a Percentage of the Nucleic Acid-Fluorophor Fluorescence Value Endogenous fluorescence

Calf Calf WF WF Sand WF WF WF

liver RNA (%) thymus DNA egg larvae lance larvae metamorphosed muscle liver

Residual fluorescence

Hoechst settings

EB settings

Hoechst + enzymes

EB + enzymes

0 0 4-5 0 2-3 13-15 26-27 12-17

0 0 8-12 0 l-2 0 19-22 4-5

0 0 80-87 1 0.4-1.6 8-11 54-56 11-34

0 0 8-11 1 l-2 2 22-26 2-4

Note. Endogenous fluorescence is fluorescence of the sample in the absence of dye. Residual Auorescence is fluorescence remaining in the presence of dye after the sample is treated with DNase and RNase.

tration and was not affected by the addition of DNase or RNase. Little or no endogenous fluorescence was observed in larval fish extracts and standards. FIA us uu. Pooled WF larvae were analyzed by FIA and the Schmidt-Thannhauser uv method (11) as modified by Munro and Fleck (12) and adapted by Buckley (1). The FIA method gave estimates of RNA and DNA concentration comparable to those obtained by the uv method, when calf liver RNA and calf thymus DNA standards were used (Fig. 4). DISCUSSION

One characteristic of the FIA method, inherent to all fluorometric methods for estimation of nucleic acids, is the sensitivity to the selection of standards. A wide range of values can be calculated from the same samples and procedure-simply by changing standards. Bentle et al. (2) reported a ratio of fluorescence of transfer RNA to CT DNA in EB of 0.21. This differs from LePecq and Paoletti’s (13) ratio of 0.46 (rat liver or yeast RNA and CT DNA) and Clemmesen’s (3) reported ratio of 0.49 (yeast RNA and CT DNA). Our data illustrate that a variety of ratios ranging from 0.199 (CL RNA and CT DNA) to 1.320 (MS 2 RNA and salmon sperm DNA) can be calculated by using the slopes of the different standards (Table 1). We chose to use calf thymus DNA and calf liver RNA as our working standards because consistent commercial preparations are readily available and this combination of standards resulted in estimates of nucleic acid concentrations comparable to those of the Buckley (1) uv method. The potential variability in results from laboratories using different procedures and nucleic acid standards requires increased attention to standardization among laboratories.

AND

BUCKLEY

Estimates of DNA contamination in the calf liver RNA standard also depend upon the DNA standard used. Contamination averaged ~1% of the spectrophotometrically determined value when calf thymus DNA standards were used and 4% when salmon sperm DNA standards were used. Bentle et al. (2) found a 4% DNA contamination in transfer RNA (Type XXI, Escherichia coli), when compared to a calf thymus DNA standard. DNA fluoresces 1.3-5~ more than RNA in EB, thus any contamination of the RNA standard by DNA will be magnified. If the extent of DNA contamination is large, the RNA standards should be cleaned up or the contamination taken into account in the calculations. Fluorescence efficiency of the nucleic acid-fluorophor complex is sensitive to temperature fluctuation (14). As room temperature increases, the fluorescence yield decreases. We found, as did Karsten and Wollenberger (15), that running the assay in an environment with at least moderate (&2’C) temperature control is essential. Nuclear proteins limit the accessibility of EB and Hoechst to DNA (16,15). Treatment with N-lauroylsarcosine, an anionic detergent, enhanced the EB fluorescence of tissue extracts. Doubling the sarcosine concentration from 1 to 2% increased the amount of nucleic acid extracted in liver and muscle tissue; however, it necessitated a 20-fold dilution of the sarcosine before analysis, to reduce fluorescence of the blank. If tissue size were not limiting, this would be the extraction concentration of choice for those tissues. Our extraction procedure requires additional injections to analyze eggs, muscle, and liver tissue to estimate the endogenous fluorescence. If the amount of tissue available were limiting, as in individual egg analyses, the samples would have to be hand injected. Since the endogenous fluorescence in this study was unchanged by the addition of RNase or DNase and was

FIG. 4.

Comparison of estimates of RNA and DNA in pooled WF larvae, obtained using the FIA or uv method. Standards used in the FIA method were CT DNA and CL RNA. The line represents a 1:l correspondence for the two methods.

FLOW

INJECTION

ANALYSIS

equal to the residual fluorescence in both dyes, the material responsible was probably an interfering substance left in the crude extract-not a nucleic acid. In Hoechst, the residual fluorescence was greater then the endogenous fluorescence in muscle and eggs. The larger residuals could be due to the incomplete digestion of the DNA by the enzymes, particularly since as few as four A-T base pairs (17,18) are required for fluorescence. The FIA method described provides a simple and rapid means of assaying RNA and DNA concentrations in tissues. The high degree of sensitivity and elimination of the homogenization step make it ideal for the analysis of large numbers of individual larval fish and other small organisms. Its application to other tissues may require additional injections to determine the endogenous fluorescence of the extract at selected excitation and emission settings. REFERENCES 1. Buckley, 2. Bentle,

116,516.

L. J. (1979) L. A., Dutta,

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Res. Board Metcoff,

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RNA

3. Clemmesen, C. M. (1988) Meeresforschung 32, 134-143. 4. Murray, M. G., and Paaren, H. E. (1986) Anal. Biochem. 154, 638-642. 5. LePecq, J.-B., and Paoletti, C. (1967) J. Mol. Biol. 27, 87-106. 6. Morgan, A. R., Lee, J. S., Pulleyblank, D. E., Murray, Evans, D. H. (1979) Nucleic Acids Res. 7,547-569. 7. Weisblum, B., and Haenssler, E. (1974) Chromosomu 260. 8. Comings,

D. E. (1975)

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14. Chen, R. F. (1967) 15. Karsten, U., and 464-470. 16. Brodie, 17. Martin, 18. Harshman, 4825-4835.

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