An ethidium bromide-agarose plate assay for the nonradioactive detection of cDNA synthesis

ANALYTICAL

BIOCHEMISTRY

178,269-272

(1989)

An Ethidium Bromide-Agarose Plate Assay for the Nonradioactive Detection of cDNA Synthesis’ Alice A. Christen

and Beverly

Montalbano

U.S. Department of Agriculture, Agricultural ResearchService, Southern RegionalResearchCenter, 1100Robert E. Lee Boulevard, New Orleans,Louisiana 70179

Received

October

11,1988 MATERIALS

Ethidium bromide was used to determine the success of cDNA synthesis reactions. Since ethidium bromide in agarose can be used to quantitate RNA and DNA, conditions under which the greater fluorescence of doublestranded DNA (dsDNA) is utilized were devised to assay dsDNA synthesis from mRNA. Ethidium bromide at 5 pg/ml in agarose allowed quantitative detection of cDNA in the range of 0.03 to 0.0015 pg. Sodium dodecyl sulfate had an adverse effect on the measurement of cDNA. Subsequent cDNA analysis by alkaline gel electrophoresis and staining in 5 pg/ml ethidium bromide allowed accurate and rapid sizing of cDNA and required only 0.1-0.05 pg cDNA. o 1989 Academic Press, IIN.

Synthesis of cDNA from mRNA is monitored by radioactive methods and gel electrophoresis. The firststrand synthesis may be monitored with 32P incorporation to determine the yield. This similarly may be followed by 3H incorporation in the second strand. In addition to trichloroacetic acid precipitation, gel electrophoresis may be used to determine the synthesis of cDNA and give a size determination (1,2). In contrast to the above procedures we have developed a method to detect nanogram amounts of cDNA synthesized. This method is based on ethidium bromide detection methods for mRNA (3) and DNA (4). Radioactive compounds are not required to determine the amount of cDNA produced because of the sensitivity of our ethidium bromide assay.

1 Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.

0003-2697/89 $3.00 Copyright 0 1989 by Academic Press, All rights of reproduction in any form

AND

METHODS

Nucleic acids. Rabbit globin poly(A)+ RNA, RNA ladder, X DNA, $X174 (+)-strand DNA (ssDNA)’ (Bethesda Research Labs), BMV RNA (Promega Biotee), 12 and 40- to 60-mer linkers (Pharmacia) were used in this study. cDNA was made from globin RNA by use of oligo(dT) primers, reverse transcriptase, RNase H, and DNA polymerase (cDNA Synthesis System, Amersham Corp.). Plate assay. Detection plates consisted of 1% agarose (FMC Corp; SeaKem HGT) in 10 mM Tris, pH 7.4, and 1 mM EDTA, pH 8.0 (TE buffer), boiled until dissolved, cooled to approximately 6O”C, and ethidium bromide, 1 to 10 pg/ml, added prior to pipetting into a 15 X loo-mm petri dish, 10 ml/dish. Plates were dried open in a 37°C incubator for 20 min, wrapped in aluminum foil, and stored at 4°C until use. Samples, with or without vacuum evaporation (Savant), were spotted, in the absence of or presence of SDS, in l-cl1 volumes onto the agarose surface. Plates were then incubated at 37°C for 20 min to allow for absorption of the liquid into the plate and for the nucleic acid to diffuse. The uncovered dish was then placed on an ultraviolet-light transilluminator, 300-nm wavelength (Fotodyne), to visualize the ethidium bromidestained nucleic acids. Photographs were taken with Polaroid Type 667 film at F 5.6 for 1 s or Type 55 film at F 5.6 for 80 s. The intensity of the fluorescence was used for the comparison of spots containing different concentrations and type of nucleic acid. Gel electrophoresis. The sizes of nucleic acids were examined by agarose gel electrophoresis with minigels (International Biotechnologies, Inc.). Agarose gels (1%) were electrophoresed in 0.1 M Tris, 0.05 M boric acid, 2Abbreviations double-stranded

used: ssDNA, &1X174 DNA, TCA, trichloroacetic

(+)-strand acid.

DNA;

dsDNA,

269 Inc. reserved.

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CHRISTEN

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FIG. 1. Quantitation of nucleic acids in ethidium bromide-agarose. The plate contains 5 @g/ml ethidium bromide in 1% agarose. BMV RNA: 0.25,0.125,0.06,0.03 pg (Row 1); 0.015,0.007,0.003 pg (Row 2); @X ssDNA: 0.25, 0.125, 0.06, 0.03 pg (Row 3); 0.015, 0.007, 0.003, 0.0015 fig (Row 4); X dsDNA: 0.3, 0.15, 0.07, 0.03 pg (Row 5); 0.015, 0.007,0.003,0.0015 pg (Row 6).

MONTALBANO

trast than 5,15, or 20 ml. The margin of the fluorescing spot was fuzzy for 12 and 60 mer linkers as opposed to the even margin of the spots produced with the other nucleic acids tested, all of which were >600 bp. The margin was also fuzzy for RQl DNase (Promega)-treated pBR322 DNA, and no fluorescence occurred with dinucleotide triphosphates. Globin RNA was used for the detection of cDNA synthesis to test the usefulness of the 5 kg/ml ethidium bromide concentration in 10 ml agarose per plate. The concentration of globin in the first-strand synthesis was 0.05 pg/pl. Samples were placed on ice until use and consisted of aliquots taken both before and after incubation of the first-strand reaction, an amount of globin equivalent to the first sample (0.05 pg), and an aliquot taken after second-strand synthesis. The fluorescence of these samples was compared to the appropriate controls (i.e., globin was compared to BMV RNA controls, first-strand synthesis to ssDNA controls, and second-strand synthesis compared to X controls). The fluorescence of globin spots (Fig. 2) was comparable to that of BMV RNA of 0.065 pg. The first-strand fluorescence was comparable to that of ssDNA of 0.015, and the second-strand comparable to that of dsDNA of 0.03 pg. Since the input RNA

0.003 M EDTA buffer. Gels were stained 15 min with 0.5, 5, or 10 pg/ml ethidium bromide followed by 15 and 45 min destaining in water. Alkaline agarose gels (1.4%) were prepared with sodium hydroxide (5). Nucleic acids loaded onto alkaline gels were treated with Geneclean (Bio 101) or Nensorb (New England Nuclear) instead of phenol to remove magnesium and concentrated by vacuum evaporation prior to resuspension in alkaline loading buffer. With Geneclean, the pellet was washed four times to completely remove any trace of sodium iodide and then vacuum-evaporated (Savant) to dryness to remove all traces of ethanol. RESULTS

BMV RNA spotted on agarose plates containing 1 pg/ ml ethidium bromide (3) produced a range of fluorescent intensities at 0.25 to 0.06 pug; however, X DNA failed to produce different intensities at 0.3 to 0.03 pg. Varying the amount of ethidium bromide in the agarose from 5 to 10 pg/ml showed that 5 pg/ml gave a range of intensities for both RNA and DNA, and detection was apparent even at 0.0075 pg RNA and 0.0015 pg DNA (Fig. 1). Amounts of 7.5 and 10 pg/ml gave too much background fluorescence, while 2.5 pg/ml did not display the sensitivity of 5 pg/ml. A lo-ml volume of ethidium bromideagarose per plate gave fluorescent spots with better con-

FIG. 2. Quantitation of globin cDNA in ethidium bromide-agarose plate assay. Row 1: BMV RNA 0.015, 0.03, 0.06, 0.125 pg; Row 2: X dsDNA 0.010,0.015,0.020,0.025,0.030 pg; Row 3: 0.05 ag globin RNA, 0.05 pg globin RNA in first-strand reaction mixture before synthesis, same volume after first strand synthesis, an equivalent amount then subjected to RNase H, an equivalent amount following second-strand synthesis; Row 4: same as Row 3 except the last three were purified with Nensorb columns; Row 5: h dsDNA 0.040, 0.035 pg (columns 4 and 5); Row 6: @X ssDNA 0.006,0.0125,0.025,0.05 pg.

NONRADIOACTIVE

DETECTION

was 0.05 pug, this indicated that synthesis of the first strand was nearly 30% successful and the second strand was the amount expected, i.e., double the first-strand amount. Because the ssDNA fluorescence is weaker than the dsDNA, this change in intensity from first- to second-strand reactions also indicates that the final product was double stranded. The globin RNA/sscDNA hybrid fluoresced equally prior to or following RNase H digestion, indicating the hybrid and ssDNA fluoresce comparably. Treatment of the first- and second-strand reaction products with Nensorb prior to plating also allowed quantitation (Fig. 2). In contrast, Geneclean left residual glass beads which interfered with quantitation (data not shown). The effect of SDS on cDNA detection was determined because SDS may be added to the stop reaction at the end of the second-strand synthesis. SDS applied to the agarose-ethidium bromide plates produced a translucent spot which increased in size with the increase in concentration (0.1, 1, 5, 10, and 20%). A slight fluorescence was present in the center of the translucent spot at 5% and was greater at 10 and 20% SDS. SDS added to BMV RNA affected the concentrations at <0.125 pg, causing an approximate twofold increase in fluorescence and an increase in spot diameter. SDS did not increase the fluorescence of X DNA, but adverse effects were apparent. Spots fluoresced unevenly and were irregular in shape, making them difficult to interpret. Agarose gel electrophoresis of globin RNA yielded a 600- to 650-bp band. Alkaline gel electrophoresis of aliquots of the first- and second-strand reactions gave bands of comparable size (Fig. 3). Genecleaning of the first-strand reaction product was minimally effective and that of the single-stranded DNA generated by RNase H digestion was ineffective (Fig. 3, Lanes 6 and 7) in visualizing products in comparison to Nensorbed or uncleaned products. Alkaline gels stained with 0.5 or 10 pg/ml ethidium bromide were less sensitive in DNA detection than those stained with 5 pg/ml. While nonalkaline agarose gels were five times more sensitive than the 0.05- to 0.1-118 amount of DNA needed for detection in alkaline gels, the sizing of cDNA was not accurate, giving a larger size band. DISCUSSION

The results demonstrate that nanogram quantities of cDNA synthesized from poly(A)+ RNA can be measured by an ethidium bromide assay. The method is simple and the following procedure is recommended: (i) prepare 1% agarose plates containing TE buffer, boil until dissolved, add 5 pg/ml ethidium bromide, pipet 10 ml into petri dish, allow to solidify at room temperature, dry fresh plates uncovered for 20 min in an incubator at 37°C and store at least 1 day wrapped in foil at 4°C since fresh

OF

cDNA

SYNTHESIS

271

FIG. 3. Qualitation of globin cDNA synthesis in alkaline agarose gels. Gels were stained with 5 pg/ml ethidium bromide following a l-h neutralization in TBE buffer. HindIII/HoeIII X dsDNA, 0.2 pg (Lane l), 0.1 fig (Lane 2); globin cDNA from second-strand reaction, estimated 0.15 bg, uncleaned (Lane 3), Nensorbed (Lane 4), Genecleaned (Lane 5); globin first-strand reaction product, Genecleaned (Lane 6); second-strand reaction without DNA polymerase, Genecleaned (Lane 7), Nensorbed (Lane 8); first-strand reaction product, Nensorbed (Lane 9), uncleaned (Lane 10).

plates may produce spots with undesirable characteristics; (ii) prepare dilution series of X DNA, 4X174 ssDNA, and BMV RNA consisting of 0.1 to 0.0015 pg/pl, e.g., amounts used in Fig. 2; (iii) collect these samples and immediately place on ice 1 ~1 (l/20 of the original reaction volume) of first-strand reaction before and after incubation, 5 ~1 of the five-fold dilution of the firststrand reaction into the second-strand reaction, after incubation, and an amount of poly(A)+ RNA equivalent to that contained in l/20 of the first-strand reaction (it may be necessary to concentrate some samples in a vacuum evaporator to 1 ~1 final volume); (iv) spot 1 ~1 of each standard and each sample on an agarose-ethidium bromide plate using a 24-place grid, place plate in a 37°C incubator for 20 min to allow the liquid to absorb and the nucleic acid to diffuse; (v) place uncovered dish on a ultraviolet-light transilluminator to visualize the ethidium bromide-stained nucleic acid, photograph with Polaroid type 55 film for 80 s at F 5.6, and compare the intensity of samples and standards to determine the amount of cDNA synthesized. The above method is easier and more precise for detecting small amounts of cDNA rather than the commonly used TCA precipitation or agarose gel electropho-

272

CHRISTEN

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resis using radioisotopes. It also avoids the need for use of radioisotopes. The method has been used successfully for monitoring the cloning of soybean and cotton cDNA, for quantitating amounts of RNA from oligo(dT) chromatography, RNA transcripts from SP6 type vectors, DNA from plasmid minipreps, and for various nucleic acid purifying procedures. When this method is combined with the described alkaline gel electrophoresis procedure, both quantitative information and qualitative information on cDNA synthesis are obtained. Although alkaline gel electrophoresis protocols list phenol extraction and precipitation for sample preparation, Nensorb worked well and was also useful in quantitating samples on the plate assay.

MONTALBANO

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