Plant Molecular Biology 5: 183-190, 1985 © 1985 Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands

A microassay for detection of D N A and R N A in small numbers of plant cells Anne Crossway & Catherine M. Houck Calgene, lnc., 1920 Fifth St., Davis, CA 95616, U.S.A.

Keywords: cauliflower mosaic virus, microtechnique, nucleic acid hybridization

Summary A microtechnique for the detection of DNA or RNA in small numbers of plant cells (I-50) has been developed using cauliflower mosaic virus (CaMV) infection of turnip as a model system. Both DNA and RNA extracted from 10 mesophyll protoplasts from CaMV-infected plants can be detected by hybridization using a radioactive probe made from cloned CaMV DNA (pCaMV 10). No hybridization above background was detected in extracts of protoplasts from uninfected plants. At least 0.15 pg (11 000 molecules) of purified pCaMVI0 DNA can be detected. This method is superior to existing 'macro' techniques for nucleic acid detection as smaller amounts of tissue are required and the detection is approximately 100-fold more sensitive.

Introduction

Materials and methods

The detection of transcription is important in screening plant tissues which have been transformed by foreign DNA. Normally, several months of tissue culture are necessary to obtain enough plant material from individual transformants to perform Southern or Northern hybridization analyses. These 'macro' analyses are limited in sensitivity to detection of several picograms of nucleic acids (1). Even dot hybridizations require thousands of Cells per sample (2, 3). In an attempt to speed up such analyses, a microtechnique was developed for detecting DNA and RNA in 1-50 plant cells. Cauliflower mosaic virus (CaMV) infected Brassica campestris ssp. rapa (turnip) was used as a model system for developing the microtechnique. Using a radioactively-labelled CaMV DNA clone as a probe, CaMV DNA or RNA can be preferentially detected in mesophyll protoplasts prepared from CaMV-infected turnip plants. No hybridization of the CaMV probe can be detected in protoplasts prepared from leaves of uninfected turnip plants. Both DNA and RNA can be detected at levels 10s cpm/#g. The probe was denatured by boiling for 5 minutes and added to the hybridization solution (prehybridization solution containing 10% dextran sulfate). Filters were hybridized at 42°C for at least 12 hours. The filters were washed at 60 ° C in 1 X SSC and 0. I% SDS for 30 minutes followed by three 30 minute washes at 60°C in 0.1 X SSC and 0.1% SDS. Autoradiography was performed using Kodak XAR film and a Dupont Cronex intensifying screen for a few hours to 3 days.

Hybridization standards Each hybridization of experimental filters was accompanied by hybridization of a filter containing standards made from known quantities of pCaMV10 DNA. These filters were made by dotting 1 #I samples of serial dilutions of purified pCaMVI0 DNA onto dry Gene Screen filters which were then baked for 2 hours in an 80°C vacuum oven and hybridized as described above.

Quantitation Autoradiographs were scanned with a Zeineh Soft Laser Scanning Densitometer Model SLT R F F from Biomed Instruments, Inc., Fullerton, CA. Integrations were done with the aid of an Apple IIe and software provided with the instrument. Several different exposures of the same filters were scanned and the peak areas were plotted vs. the known quantity of DNA in the hybridization standard dots. The linear regions of these calibration plots were used to determine the amount of hybridizing nucleic acid in the experimental dots by interpolation.

Results

Several different hybridization membranes were tested using standard dilutions of plasmid DNA.

The sensitivity of the hybridization using nitrocellulose (Schleicher and Schuell BA85), Gene Screen (NEN), and Gene Screen Plus (NEN) filters were compared. The hybridization signal using nitrocellulose filters was not as strong as the Gene Screen signal. The Gene Screen Plus hybridization (which required a slightly different hybridization protocol recommended by the vendor) had greatly increased background which did not allow the relatively long (3 day) exposures necessary for the detection of signals from small amounts of nucleic acid. In addition, drops deposited on dry Gene Screen Plus tended to distort due to nonuniformity of the paper surface. Although nucleic acid binding to Gene Screen has been reported to be efficient in low salt, we tested the hybridization intensity of RNA samples bound to Gene Screen under a variety of salt conditions in an attempt to increase the binding efficiency. Serial dilutions of total RNA from CaMV-infected turnip leaves (gift from C. K. Shewmaker) were dotted onto dry Gene Screen, Gene Screen moistened with 20 X SSC, and Gene Screen which was first moistened with 20 X SSC and then dried. In each case the intensity of the hybridization signal was unchanged by the treatment. As little as 0.2 ng of total RNA could be detected after a 17 day exposure. When this RNA sample was treated with DNase I under conditions identical to the treatments for the microassay the signal was decreased by 2- to 3-fold. We do not know if this is due to DNA contamination of the RNA preparation or to RNase contamination of the DNase preparation. In addition, different methods of denaturing the DNA standards were compared. The dilutions were either made into distilled water, 0.5 M NaOH, or into 50% formamide. The samples were dotted onto Gene Screen, baked, and hybridized as usual (see Materials and methods). The intensity of hybridization to the formamide denatured DNA was 25 times the intensity of the undenatured sample and the NaOH treatment increased the hybridization signal two-fold over the formamide signal (data not shown). Because the NaOH denaturation is inconsistent with RNA detection, the protoplast extracts were denatured with formamide (see below). Variability is expected to occur in protoplasts made from different plants whose growth conditions may not be identical. Some variability might also be expected in very small samples taken from the same protoplast preparation due to differences

187 Fig. 2. Typical filtersafter hybridization and autoradiography. Preparation of hybridization standard (A) and protoplast extraction filters (B-G) is described in Materials and methods. The filter pairs BC, DE, and FG represent replica experiments performed on different days with different protoplast preparations. A) A standard dilution series of alkaline denatured (0.5 M NaOH) pCaMV l0 DNA. One #1of each dilution was dotted onto Gene Screen. Dot I. 3 000 pg; Dot 2.300 pg; Dot 3.30 pg; Dot 4. 3.0 pg; Dot 5.0.30 pg; Dot 6.0.15 pg. B, D, F) A series of four dots, each made from ten protoplasts from uninfected leaves of B. campestris ssp. rapa. Dot 1. Standard treatment. Dot 2. Standard treatment plus RNase. Dot 3. Standard treatment plus formamide. Dot 4. Standard treatment plus DNase and formamide. C, E, G) A series of four dots, each made from ten protoplasts from CaMV-infected leaves of B. campestris ssp. rapa. Dot 1. Standard treatment. Dot 2. Standard treatment plus RNase. Dot 3. Standard treatment plus formamide. Dot 4. Standard treatment plus DNase and formamide.

in l o c a t i o n o f cells t h r o u g h o u t the leaf a n d variation in viral r e p l i c a t i o n a n d t r a n s c r i p t i o n f r o m cell to cell. P r o t o p l a s t p r e p a r a t i o n s were m a d e f r o m u n i n fected o r C a M V - i n f e c t e d t u r n i p leaves as d e s c r i b e d in M a t e r i a l s a n d m e t h o d s . D r o p s c o n t a i n i n g 10 p r o t o p l a s t s each were t r e a t e d as d e s c r i b e d a n d s h o w n in Fig. 1. F i g u r e 2 shows a typical series o f

filters following e x t r a c t i o n , h y b r i d i z a t i o n , and aut o r a d i o g r a p h y . The replica filters shown in Fig. 2 were m a d e on different d a y s using different p r o t o plast p r e p a r a t i o n s m a d e f r o m infected or uninfected leaves. These replicates show the m a x i m u m extent o f v a r i a b i l i t y t h a t we o b s e r v e d f r o m one p r o t o p l a s t p r e p a r a t i o n to a n o t h e r a n d b e t w e e n different viral infections. In e x p e r i m e n t s c o m p a r i n g s u b s a m p l e s o f p r o t o p l a s t s p r e p a r e d f r o m the same p l a n t ( d a t a not shown), the v a r i a t i o n was no less t h a n o b s e r v e d f r o m p l a n t to plant. Filters B, D, a n d F are replica filters that were m a d e using uninfected cells a n d n o n e of the d o t s s h o w d e t e c t a b l e h y b r i d i z a t i o n to the p C a M V 1 0 p r o b e . Filters C, E, a n d G a r e replica filters t h a t were m a d e using C a M V - i n fected cells a n d s h o w h y b r i d i z a t i o n to the p C a M V ! 0 probe. T h e q u a n t i t y o f nucleic acid e x t r a c t e d f r o m each d r o p was e s t i m a t e d by c o m p a r i s o n of the h y b r i d i z a t i o n intensity with s t a n d a r d d i l u t i o n s of d e n a t u r e d p C a M V I 0 D N A ( F i l t e r A). D o t 1 on each of the o t h e r filters (Fig. 2) received the s t a n d a r d t r e a t m e n t with no nuclease or f o r m a m i d e steps. H y b r i d i z a t i o n of these d o t s is e x p e c t e d to result f r o m D N A or R N A that lacks significant s e c o n d a r y s t r u c t u r e or D N A t h a t has been p a r t i a l l y d e n a t u r e d d u r i n g the e x t r a c t i o n o r b a k i n g p r o c e d u r e . T h e s e c o n d d o t on each filter (Fig. 2) received the s t a n d a r d t r e a t m e n t plus R N a s e . H y b r i d i z a t i o n here is e x p e c t e d to result o n l y f r o m D N A which is singles t r a n d e d or p a r t i a l l y d e n a t u r e d either in the cell or as a result o f the t r e a t m e n t . T h e third d o t o f each set

188 (Fig. 2) received the standard treatment plus formamide under conditions (42°C, 50% formamide) that might be expected to disrupt short stretches of secondary structure in the single stranded R N A molecules and partially denature the D N A upon baking. The fourth dot of each set (Fig. 2) received the standard treatment plus DNase, then was denatured with formamide. This treatment is expected to remove any D N A and to denature short stretches of secondary structure in the remaining RNA. The filters were baked, hybridized, washed, and subjected to autoradiography as described in Materials and methods. The peak areas were determined by scanning the autoradiographs with a densitometer and converted to picograms by comparison with the standard filter as described above. The results are summarized in Table I. The hybridization intensities are fairly consistent within an experiment (for example, dot 3 is always more intense than dot l which is always more intense than dot 2 or 4). However, the overall intensity does vary somewhat from experiment to experiment (in this case, Filter C hybridizations are more intense than Filters E or G). This is not too surprising because these results are obtained using cells from plants infected by a virus. The amount of viral nucleic acid might be expected to vary somewhat in cells from different infected plants, and in different protoplast preparations which could have significant variations in their metabolic activities. Table 1. Quantitation (pg nucleic acid/dot) of hybridization signals from replica filters. Dot number b

Picograms nucleic acid per dot ± S.D. in experimental filter a C

1 2 3 4

E

G

0.97+0.19 0.43+0.09 0.15+0.13 0.44 + 0.08 0.12 ± 0.03 0.06 ± 0.02 3.21 + 0.66 1.75 + 0.38 1.41 ± 0.41 0.10 ± 0.02 0.11 ± 0.04

Average pgs nucleic acid per dot ± S.D. c 0.5 0.2 2.1 0.10

±0.4 ± 0.2 ± 0.9 ± 0.04

a The signal intensity (peak area) of dots from 5 hour, 24 hour, and 3 day autoradiographs were determined using a scanning densitometer as described in Materials and methods and converted to pg by comparison with a standard curve constructed using the hybridization standards of Filter A (Fig. 2). The values are the average of determinations using all exposures which gave linear standard curves in the range of the dot intensity. b Description of experimental treatment for each dot is given in Fig. 2 legend. c The results of the three replica experiments were averaged.

Discussion We have developed a relatively rapid, reproducible procedure for the detection of specific D N A or R N A sequences in very small numbers of plant cells. During the development work on this project, twelve sets of experiments were performed in which we could always detect a nucleic acid signal from five or more CaMV-infected protoplasts. In some cases we could even detect a signal from a single CaMV-infected protoplast. The data shown in Fig. 2 and Table 1 can be analyzed in terms of the known quantities of CaMV D N A and R N A in CaMV-infected tissue. The average intensity of the signal of dot l from filters C, E, and G is approximately the same as the signal f r o m 0.5 pg of purified plasmid D N A (see Table l). If the hybridization were all due to C a M V D N A ( M W :- 5.3 )< l06 daltons), this would correspond to 57 000 D N A molecules or 5 700 D N A molecules per cell. If the hybridization were all due to C a M V R N A which consists primarily of two transcripts, 8.2 kb and 1.9 kb (7), average M W -- 1.7 × l06 daltons, this would correspond to 180 000 molecules or 18 000 molecules per cell. Because this drop received no formamide denaturation, these are expected to be underestimates of the total amount of D N A or RNA. Dot 2 has a signal intensity (Filters C, E, and G, Fig. 2) which corresponds to approximately 0.2 pg or 2 300 D N A molecules per cell. As this dot contains only DNA, this figure should be compared with the estimated 1 000 000 C a M V viral particles (10 pg) found in an infected cell (R. Gardner, personal communication). This dot received no denaturing treatment so it may be that this small amount of hybridization results from partially single stranded D N A molecules that are present in infected cells (8). It has been postulated that such structures might occur in the course of viral replication

(9). The intensity of hybridization in dot 3 (Filters C, E, and G, Fig. 2) corresponds to approximately 2. l pg which would be the equivalent of 24 000 molecules per cell of D N A or 76 000 molecules per cell of RNA. Comparing this result to dot I shows that the hybridization signal has increased approximately four-fold due to this denaturation step. However, the levels are still approximately 40-fold lower than would be expected from the known quantities of

189 viral DNA in the infected cells (see above). This could either be because the DNA is not completely denatured or because DNA in viral inclusion bodies is not extracted well by this method. Lack of complete denaturation is unlikely to account for more than a two-fold difference based on results with DNA standards (see Results). In addition, similar hybridization intensities were obtained when dots which had received the standard treatment were denatured in 0.5 M N a O H for one hour at room temperature (data not shown). It has been reported that DNA from viral inclusion bodies is difficult to extract in a total cellular DNA preparation (see, for example, 8) and it is possible that the DNA from viral inclusion bodies is not all available for hybridization using our technique. The hybridization intensity of dot 4 (Filters C, E, and G, Fig. 2) corresponds to approximately 0.1 pg. This dot contains only RNA, so this signal is equivalent to 3 600 molecules of RNA per cell. This value is close to the estimated number of CaMV RNA molecules (2 000) expected in an infected cell (calculated assuming 2% of the polyadenylated mRNA in an infected cell is from CaMV, see 7). The hybridization intensities vary somewhat between the replicate filters (compare filters C, E, and G in Fig. 2). This is not unexpected and three possible explanations can be proposed. First, the very small sample size (10 cells) means that variations in the size or metabolic activity of the individual protoplasts selected for analysis could result in differences in the quantity or in the conformation of the viral nucleic acids in different drops. Also, the course of the viral infection in a particular plant and environment may affect the amount of viral nucleic acid present in cells. Experiments described in the Results section showed considerable variation in the hybridization signal not only from plant to plant but from cell to cell within a plant. Second, the amount of denaturation of the nucleic acids under our conditions could be variable. Third, it is possible that non-specific nuclease activity in the DNase preparation is sufficient to partially degrade the small quantity of RNA we are trying to detect. This possibility was tested by DNase treatment of standard dilution series of CaMV RNA (data not shown) which showed a reduction in the hybridization signal after nuclease treatment. Increasing the number of cells per microdrop overcomes this nuclease problem (up to 50 cells per drop have been used successfully here). With the aim of detecting

and quantitating transient expression following microinjection of foreign DNA, we are currently experimenting with other denaturing conditions and other methods of sample preparation not requiring nucleases to make the RNA detection more reproducible for 1-10 cells per drop. Edstrom (10) and Scalenghe et aL (11) described a microextraction technique using hanging drops. The present technique, using lying drops, has the advantage that the chloroform wash does not fall away from the drop so it continues to disperse the phenol layer for an extended period of time. The necessity for using volumetric pipettes to measure the quantities of chemicals added and for repeated phenol extractions (see 10) was not realized here; estimation by observation of drop expansion appears to be adequate. Currently, this technique is being adapted to the study of DNA stability and transcriptional activity following microinjection of foreign DNA. Such studies would involve the detection of a relatively large number of nucleic acid molecules per cell, as is described here. Detection of transformation, in which 1-50 copies of foreign DNA per genome are stably integrated and are transcribed at relatively low levels, would require even greater sensitivity than obtained here to be usable with small numbers of cells. Some modifications will be attempted to improve upon the sensitivity of the present technique. There are other potential applications of this technology. As shown here with the CaMV model system, the technique may be useful for the maintenance of virus,free stocks during micro-propagation. It could also be used to study the life cycle of viruses in conjunction with microinjection techniques. Fluorescent antibody techniques can identify single infected cells. However, the present report is, to our knowledge, the first demonstration of detection of viral nucleic acids in single plant cells.

Acknowledgements The authors wish to thank Dr Maurice Moloney for the preparation of Brassica protoplasts used in these experiments, Ms Julie Pear for valuable assistance with hybridization experiments, and Dr Thomas E. Wagner for helpful advice on microtechnique. Thanks is also given to Drs Richard C. Gardner, Robert M. Goodman, Robert J. Shepherd, and Christine K. Shewmaker for critical review of the manuscript.

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References I. Thomas PS: Proc Natl Acad Sci USA 77:5201-5205, 1980. 2. White BA, Bancroft FC: J Biol Chem 257:8569-8572, 1982. 3. Manzari V, Gallo RC, Franchini G, Westin E, CeccheriniNelli L, Popovic M, Wong-Staal F: Proc Natl Acad Sci USA 80:11-15, 1983. 4. Howarth A J, Gardner RC, Messing J, Shepherd R J: Virology 112:678-685, 1981. 5. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning, Cold Spring Harbor Laboratory, 309 if, 1982. 6. Gardner RC, Howarth AJ, Hahn P, Brown-Luedi M, Shep-

herd R J, Messing J: Nucleic Acids Res 9:2871-2887, 1981. 7. Shewmaker CK, Caton JR, Houck CM, Gardner RC: Virology 140:281-288, 1985. 8. Hull R, Covey SN: Nucleic Acids Res 11:1881-1895, 1983. 9. Hull R, Covey SN: Trends in Biochem Sci 88:19-121, 1983. 10. Edstrom JE: In: Prescott DM (ed) Methods in cell physiology. Academic Press, New York, 1964, pp 417-447. II. Scalenghe F, Turco E, Edstrom JE, Pirrotta V, Melli M: Chromosoma (Bed) 82:205-216, 1981. Received 26 March 1985; in revised form 30 May 1985; accepted I I June 1985.

A microassay for detection of DNA and RNA in small numbers of plant cells.

A microtechnique for the detection of DNA or RNA in small numbers of plant cells (1-50) has been developed using cauliflower mosaic virus (CaMV) infec...
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