Journal of Histochemistry and Cytochemistry, Vol. 45, 721-728, Copyright © 1997 by The Histochemical Society, Inc.


TECHNICAL NOTE

Improved In Situ Hybridization to HIV with RNA Probes Derived from PCR Products

Richard W. Conea and Erika Schlaepfera
a Division of Infectious Diseases, Department of Internal Medicine, University Hospital, Zurich, Switzerland

Correspondence to: Richard W. Cone, University Hospital, Div. of Infectious Diseases, Dept. of Internal Medicine, Ramistr. 100, CH-8091 Zurich, Switzerland.


  Summary
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

These experiments tested the hypothesis that a pool of PCR-derived RNA probes with defined length and even representation of the target sequences could produce more specific and intense in situ hybridization signals than randomly size-reduced, plasmid-derived RNA probes. In situ hybridization was performed with sense and anti-sense HIV-1 RNA probes that were derived from PCR products tailed with the T7 RNA polymerase promoter or from plasmid DNA. In situ hybridization using a pool of seven anti-sense or sense PCR-derived RNA probes (1805 nucleotides of HIV sequence, 257 nucleotides average probe length) was compared with hybridization using anti-sense or sense RNA probes made from a plasmid representing the HIV-1 env gene (3151 nucleotides of HIV-1 target). The pooled PCR-derived probes resulted in stronger in situ hybridization signals and less background than those produced with plasmid-derived RNA probes. This method for creating PCR-derived RNA probes improves the feasibility of synthesizing multiple, discrete RNA probes for studies of specific mRNA expression because it does not require the subcloning steps used to construct plasmids. PCR-derived RNA probes may provide a viable alternative to the use of plasmid-derived RNA probes for in situ hybridization. (J Histochem Cytochem 45:721-727, 1997)

Key Words: in situ hybridization, T7 RNA polymerase promoter, RNA, human immunodeficiency virus (HIV)


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

In situ hybridization can be done with several kinds of labeled nucleic acid probes, including synthesized DNA oligonucleotides (Maxam and Gilbert 1980 ), DNA fragments generated by nick-translation (Rigby et al. 1977 ) or random priming (Feinberg and Vogelstein 1983 ), longer DNA probes made with PCR (Celeda et al. 1992 ), and RNA probes derived from DNA sequences containing 5-prime T7, SP6, and T3 RNA polymerase promoters (Cox et al. 1984 ). Each system has unique advantages and disadvantages. RNA probes often provide the best results for in situ hybridization to viral nucleic acids because they have high specific activities and are single-stranded, enabling separate detection of sense and anti-sense targets.

The standard approach for generating RNA probes requires subcloning of the sequence of interest so that an RNA polymerase promoter resides at either end of the insert. The resulting plasmid is linearized by restriction digestion and incubated with RNA polymerase to generate labeled RNA probes representing the insert. Efficient penetration of the probe into cells requires that the RNA probes have a defined length of 200-600 bases (Singer et al. 1986 ). To achieve this, longer RNA fragments must be degraded after synthesis, producing a poorly controlled range of probe lengths. Therefore, some of the labeled RNA may be too long or too short for efficient hybridization. Alternatively, shorter subclones of ideal length can be created to generate corresponding RNA probes that do not require degradation. However, this approach requires multiple subcloning procedures to generate a mixture of probes with sufficient complexity for sensitive in situ hybridization. The time-consuming process of subcloning can therefore limit the feasibility of generating multiple RNA probes.

Bacteriophage promoters (T7, T3, SP6) have been added to one PCR primer, generating a PCR product that can be used for in vitro transcription (Stoflet et al. 1988 ). Similarly, RNA probes can be made from the opposite DNA strand by attaching the promoter to the other primer. An RNA probe synthesized from a PCR-derived substrate has been used for in situ hybridization to murine serum amyloid mRNA (Young et al. 1991 ). This technique was modified and extended here to include a cocktail of HIV-directed RNA probes.

We have used a PCR-based system for RNA probe generation that results in well-defined probes without the need for subcloning. This method has been applied to detect HIV RNA in cultured 8E5 cells, a cell line that is persistently infected with HIV-1 strain BRU and has variable expression of HIV RNA.


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

PCR Generation of T7 Promoter-tailed HIV DNA
This step synthesized seven specific HIV DNA sequences (A through G, Figure 1) from an HIV-1BRU template (Folks et al. 1986 ), creating 14 PCR products. Seven PCR products representing sequences A to G had a T7 RNA polymerase promoter at the 5-prime end of the anti-sense strand (makes anti-sense RNA probe) and seven products, again representing sequences A to G, had a T7 promoter at the 5-prime end of the sense strand (makes sense RNA probe) (Table 1 and Table 2). Because each PCR product was individually transcribed to produce an RNA probe, it was not necessary to have different promoters, such as T3 and SP6, for the sense and anti-sense strands. The HIV primer sequences from primer set A were previously reported (Piatak et al. 1993 ). The HIV primer sequences for primer sets B through G were selected from HIV-1 strain BRU sequence (Wain-Hobson et al. 1985 ) using OLIGO Primer Analysis Software version 5.0 (National Biosciences; Plymouth, MN).



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Figure 1. Top line: representation of the HIV-1 genome with major genes shown as boxes; second line: scale of genome in kilobases (kb); third line: genomic locations of PCR-derived RNA probes A-G (short thick lines); fourth line: genomic location of env plasmid RNA probe (long thick line).


 
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Table 1. Primers used for generation of T7 promoter-tailed PCR products


 
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Table 2. PCR primer pairs, product orientation, product size and length of hybridizable HIV-homologous sequence in the resulting RNA probe

To reduce the cost of synthesizing 14 different PCR primers with complete T7 promoter tails, the DNA templates were generated with two successive PCR reactions (Horton et al. 1990 ). In the first PCR reaction, 14 separate amplifications were done using the primer pairs listed in Table 2. For each primer pair, one primer had no additional nucleotides (HIV-1 sequence only) and the other primer was tailed with 16 bases representing the 3-prime end of the T7 promoter ((Milligan et al. 1987 ) (Table 1). Amplifications (100 µl) contained 5 x 1013 molecules (83 pmoles) of each primer (Midland Certified Reagent; Midland, TX), 50 µl 2 x PCR Master Mix (Boehringer, Mannheim, Germany; final concentrations 200 µM each dNTP, 2.5 U Taq polymerase, 0.005% Brij35, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2) and 0.54 µg (approximately 105 copies) purified, heat-denatured 8E5 DNA as template. The 8E5 cell line carries one integrated HIV-1BRU genome per cell (Folks et al. 1986 ). The cycling conditions (Perkin Elmer 9600 Thermal Cycler) were 97C for 1 min, five cycles of 96C for 30 sec, 45C for 90 sec, 72C for 30 sec, 35 cycles of 96C for 30 sec, 50C for 30 sec, 72C for 30 sec, ending with 72C for 5 min. The 14 reaction products were electrophoresed in 3% NuSieve agarose (FMC Bioproducts; Rockland, ME) gels, stained with ethidium bromide in water, and destained in 10 mM Tris-HCl, pH 8. The expected bands were excised from the gels, dissolved in 1 ml 10 mM Tris-HCl, pH 8, at 95C for 5 min, and diluted 1:100 in prewarmed TE (10 mM Tris-HCl, pH 8, 1 mM EDTA).

In the second reaction, the diluted amplification products from the first reaction were reamplified to extend and complete the full-length 23-BP T7 promoter. Each of the 14 amplifications contained one HIV-1 primer (HIV sequence only) as used in the first round of PCR and a second "universal" T7 primer (T7U; Table 1) containing the remaining seven nucleotides of the T7 promoter and an additional seven nucleotides at the 5-prime end to improve T7 polymerase efficiency (Kain et al. 1991 ). The terminal seven nucleotides also introduced a HindIII restriction site, although the HindIII site was not used in these studies. Table 2 lists the primer pairs in the second round of PCR. After the first round of PCR, we found that Perkin Elmer's AmpliTaq polymerase and GeneAmp 10 x PCR buffer performed better than the Boehringer Mannheim Taq polymerase and PCR Master Mix used for the first round of PCR. Each primer pair was individually optimized with respect to Mg concentration, denaturation temperature, and annealing temperature. The following represents a consensus protocol for all 14 PCRs. Amplification reactions (100 µl) contained 5 x 1013 molecules (83 pmoles) of each primer, 250 µM each dNTP (Pharmacia Biotech; Piscataway, NJ), 10 µl 10 x GeneAmp PCR buffer [500 mM KCl, 100 mM Tris-HCl, pH 8.3, 15 mM MgCl2, 0.1% gelatin (w/v); Perkin Elmer], 0.5 µl (2.5 U) AmpliTaq polymerase (Perkin Elmer), and 1 µl of diluted PCR products from the first round of PCR. The amplification conditions were: 96C for 1 min, 40 cycles of 96C for 30 sec, 52C for 1 min, 72C for 30 sec, ending with 72C for 5 min. The PCR products were electrophoresed in 3% NuSieve agarose gels and the resulting bands were excised and purified using the Qiaex II Gel Extraction Kit (Qiagen AG; Berne, Switzerland).

35S-Labeled RNA Probes from PCR Products
Fourteen RNA probes (seven sense, seven anti-sense) were separately transcribed and labeled in a reaction containing the respective PCR product (0.2 µg), T7 polymerase, and 35S-labeled UTP using the RNA Transcription Kit (Stratagene; Zurich, Switzerland) according to the manufacturer's instructions. The resulting RNA probes were digested with DNAse to remove template DNA and purified by column chromatography (Quick-Spin G-50 columns; Boehringer Mannheim). Specific activities of the RNA probes ranged from 1 x 108 to 3.45 x 108 cpm/µg RNA. RNA probe lengths were documented by agarose gel electrophoresis and autoradiography.

Mixtures of sense or anti-sense RNA probes A through G were made by combining equal numbers of cpm from each probe. The resulting RNA probes had an average HIV-homologous length of 257 bases. When all seven RNA probes were used together, they represented 1805 bases of HIV (Table 2).

Plasmid-derived RNA Probe
A plasmid containing a 3151-BP insert representing the HIV-1 env gene and flanking T3 (anti-sense) and T7 (sense) promoters was used as a positive control for generating RNA probes (kindly provided by Dr. Volker Adams). The plasmid was linearized by EcoR1 or Xho1 restriction enzymes to create anti-sense and sense RNA polymerase templates, respectively. One µg of linearized plasmid was used in the 35S-labeling reaction, followed by DNAse digestion, alkaline hydrolysis size reduction, phenol/chloroform extraction, and ethanol precipitation. The average probe lengths were 100-600 nucleotides, as documented by agarose gel electrophoresis and autoradiography.

In Situ Hybridization
Cultured 8E5 cells were harvested by centrifugation, washed, and resuspended in PBS to 1 x 107 cells/ml. Cell aliquots of 10 µl (100,000 cells) were spotted onto Super Frost Plus coated glass slides (Huber & Co; Reinach, Switzerland, or Merck, Darmstadt, Germany), fixed in 4% paraformaldehyde-PBS for 20 min, rinsed in PBS, successively dehydrated in 60%, 80%, 90%, and 100% ethanol, air dried, and stored in individual heat-sealed plastic bags (Folienschlauch, 360 x 0.1 mm, no. A361001; Audion Elektro, Klere, Germany) at -70C until needed.

Before hybridization, the slides were defrosted, rehydrated in graded ethanols, refixed in 4% paraformaldehyde-PBS, soaked (10 min) in 0.1 M HCl, rinsed, digested with Proteinase K (1 µg/ml for 20 min), rinsed in PBS-0.2% glycine, fixed again in 4% paraformaldehyde, soaked again in PBS-0.2% glycine, soaked in TEA (0.1 M) with two additions of acetanhydride, rinsed in PBS, dehydrated in graded ethanols, and air-dried.

For hybridization, the cell spots were overlaid with 20 µl hybridization solution (50% formamide, 10% dextran sulfate, 2 x SSC, 200 ng/µl tRNA, 1 U/µl RNAsin, 25 mM DTT, 1:100 Denhardt's solution) containing 1, 2, 4, or 6 million cpm 35S-labeled RNA probe, coverslipped, and incubated at 52C overnight. The PCR-derived RNA probes were used individually or mixed. After hybridization, the slides were washed two times in 50% formamide, 2 x SSC, 1 mM EDTA, 10 mM DTT at 52C for 20 min each wash, then rinsed in 2 x SSC, 10 mM DTT at 52C. The slides were treated with RNAse (12.5 U/ml RNAse A, 10 µg/ml T1 RNAse, 2 x SSC) for 30 min at 37C, washed in 2 x SSC (15 min, 50C), dehydrated in graded ethanols, and air-dried.

Hybridized probe was detected by autoradiography using Hypercoat emulsion (Amersham International; Poole, UK) according to the manufacturer's instructions. The coated slides were exposed for 4 days before developing and fixing the emulsion. Cells were lightly counterstained with hematoxylin.

The hybridization results were quantitated by counting the numbers of strongly positive cells (entire cell obscured by contiguous silver grains), all positive cells, and all negative cells in the anti-sense hybridizations. Similarly, the amount of background was determined by counting silver grains in the sense hybridizations. Counting was performed in 25 locations per slide, representing a total of 0.01 mm2, for each hybridization condition, using a calibrated ocular reticule and x 100 magnification. The observer was blinded regarding the experimental conditions for each slide, and duplicate counts confirmed reproducibility. Comparisons between hybridization conditions were evaluated with the two-tailed Students' t-test for non-paired observations using Microsoft Excel 4.0 software.


  Results
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Materials and Methods
Results
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Hybridization with Individual PCR-derived RNA Probes
The hybridization signals from each PCR-derived anti-sense and sense RNA probe (A-G) were separately assessed. Each individual RNA probe (106 cpm) was applied in a separate hybridization to 8E5 cells. HIV RNA was successfully detected with each individual probe, although the signal intensities varied among probes. Probes E and F produced very strong signals, probes A, D, and G produced strong signals, probe C produced a moderate signal, and probe B produced a weak signal (data not shown).

Signal Intensities with PCR Probe Mix and Plasmid-derived Probe
The sensitivity of in situ hybridization with a mixture of seven PCR-derived RNA probes (PCR-derived probe mix) was compared to the sensitivity when the plasmid-derived RNA probe was used. 8E5 cells were hybridized with 1 x 106, 2 x 106, 4 x 106, and 6 x 106 cpm of PCR probe mix or equivalent amounts of plasmid-derived probe. No positive cells were observed in hybridizations with sense probes, which served as negative controls. The data from this experiment are illustrated in Figure 2.



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Figure 2. Photomicrographs of hybridized 8E5 cells. The hybridization signal is seen as black silver grains. Hybridization with antisense (A-D) and sense (E-H) plasmid-derived RNA probes from a 3.1-KB fragment of the HIV-1 env gene; hybridization with anti-sense (I-L) and sense (M-P) mixture of seven PCR-derived RNA probes representing 1.8 KB of target sequence. From left to right, the columns show hybridizations done with increasing amounts of RNA probes, as indicated at the top of each column. Cells in the anti-sense pictures appear larger than cells in the sense pictures because (a) the focal plane in the anti-sense pictures was set for clarity of the silver grains, making the cell edges unclear, (b) silver grains extend beyond the cell edges, making positive cells appear larger, and (c) the focal plane in the sense pictures was set for clarity of the cells, clearly defining the cell edges so that they appear smaller than in the anti-sense pictures. Bar = 50 µm.

The proportion of strongly positive cells (strongly positive cells/moderately positive + weakly positive cells) was used as a measure of signal intensity (Figure 3a). The anti-sense PCR-derived probe mix produced a higher proportion of intensely reactive cells (0.52) than the anti-sense plasmid-derived probe mix (0.20) at the highest probe concentration, 6 x 106 cpm per slide (p=0.002, t-test). For the lower probe concentrations (1 x 106, 2 x 106, 4 x 106 cpm), the proportions of strongly positive cells were not significantly different between the two probe types (p = 0.645, 0.345, 0.380, respectively). The proportion of strongly positive cells with the plasmid-derived probe reached a maximum (0.30) at 4 x 106 cpm probe concentration and the signal intensity did not improve when more probe was added (Figure 3a). In contrast, the proportion of strongly positive cells with the PCR-derived probe mix continued to increase as the probe concentration increased. However, the percent of positive cells (all positive cells/positive + negative cells) was not significantly different between the two probe types at each probe concentration (p>0.155 for all comparisons).



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Figure 3. The signal intensities (a) and background silver grains (b) were evaluated at each of the four probe concentrations (horizontal axes) for both probe types, PCR-derived probe mix (solid lines, filled diamonds) and plasmid-derived probe (dashed lines, filled squares). (a) The proportion of positive cells with strong signal intensity (vertical axis) steadily increased with increasing PCR-derived probe concentration. The proportion of strongly positive cells with plasmid-derived probe reached a lower maximum and did not improve when more probe was added. (b) The background silver grains per 0.01 mm2 (vertical axis) with sense probes increased almost eightfold with a sixfold increase in plasmid-derived probe concentration, whereas a similar increase in the PCR-derived probe concentration produced little added background.

Background with PCR Probe Mix and Plasmid-derived Probe
The plasmid-derived probe produced more background silver grains than the PCR-derived probe mix, as determined by counting silver grains in the negative control slides hybridized with sense probes (Figure 2E-H and Figure 2M-P, respectively). With the plasmid-derived probe, the background increased steadily as the total amount of probe increased, resulting in almost eightfold more grains at the highest probe concentration than the lowest (Figure 2b). The PCR-derived probe mix showed only slightly more silver grains at the highest compared with the lowest probe concentrations. Also, the PCR-derived probe produced significantly less background than the plasmid-derived probe at the two highest probe concentrations, 1 x 104 and 1 x 106 cpm (p<0.001 for both comparisons).


  Discussion
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Materials and Methods
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The RNA probes from PCR-derived HIV DNA products provided higher signal and less background than equivalent probes derived from larger pieces of cloned HIV DNA. The superior signal intensity and reduced background of the PCR-derived RNA probes is especially striking when the relative complexity of the two probe systems is considered: The 1805 bases of hybridizable sequence represented in the PCR-derived probe mix are only 58% of the 3100 bases represented in the plasmid-derived RNA probe.

The signal improved steadily as increasing amounts of the PCR-derived probe mix were added, with a maximum at the highest concentration tested, 6 x 106 cpm applied probe (Figure 2a). In contrast, the plasmid-derived probe reached a maximal signal between 2 x 106 and 4 x 106 cpm probe. Furthermore, background from the plasmid-derived probe increased steadily with increasing probe concentration, suggesting that further escalation of probe concentration would not result in improved performance. This reaffirms the practice of keeping plasmid-derived probe concentrations low (Komminoth et al. 1992 ). Reduced background and improved signal intensity with the PCR-derived probe mix suggest that the upper limit of probe concentration may be extended with this method, leading to better hybridization signals.

We hypothesize several factors that could account for improved signal from PCR-derived RNA probes. First, random size reduction of larger plasmid-derived RNA probes produces a wide range of RNA probe lengths, some of which are probably either too long or too short for effective in situ hybridization. Therefore, a subset of the plasmid-derived probes may hybridize nonspecifically or inefficiently when the hybridization conditions are optimized for the average probe length. In contrast, the PCR-derived RNA probes have similar lengths that were designed to optimize probe penetration and hybridization in one hybridization condition. Second, RNA polymerase does not always complete every strand extension when long templates of greater than 500 bases are used (Milligan and Uhlenbeck 1989 ). As a result, RNA near the promoter of a long plasmid template is quantitatively more represented than RNA distant from the promoter. When the PCR-derived probes are mixed in equal proportions, the entire hybridizable region is more evenly represented. Third, unsuitably small or large RNA segments created by random size reduction may contribute to background. Washing conditions that are optimized for specific probe lengths represented in the PCR-derived probes could be more effective in removing non-specific hybridization.

In spite of increased signal intensity with the PCR-derived probes, the absolute numbers of positive cells were not significantly different between the two probe types. This was surprising because we expected that increased signal would reveal weakly positive cells that might be otherwise be missed. One possible explanation relates to the cell type used for these studies: All of the cultured cells were infected. The high prevalence of positive cells may have masked subtle increases in weakly positive cells. Alternatively, the increased signal from PCR-derived probes may apply more to strongly positive cells than to weakly positive ones. Further studies with other cell types, such as tissue sections that have only a few positive cells, may help to clarify this point.

The differences in hybridization with each individual PCR-derived RNA probe may be due in part to the presence of both spliced and unspliced HIV RNA in the 8E5 cells (Schwartz et al. 1990 ). Probes D through G, which gave either strong or very strong signals, hybridize to the HIV env gene (Figure 1). The env gene can be transcribed in some spliced HIV mRNA species, so that the signal represents a subset of the spliced HIV RNA as well as the full-length genomic HIV RNA (Purcell and Martin 1993 ). However, this hypothesis does not explain why probe C, also homologous to the env gene, produced only a moderate signal. Furthermore, probe A, which hybridizes to the gag gene and should represent mostly unspliced HIV RNA, gave a strong hybridization signal. In contrast, probe B, which hybridizes to the pol gene, gave the weakest signal. It is possible that secondary structure of the RNA probes or targets plays a role in the differing hybridization signals, suggesting that further optimization of the hybridization protocol might improve the signals from probes B and C.

In conclusion, we have demonstrated that PCR-derived template DNA allows rapid production of highly efficient and specific HIV RNA probes. This technique allows researchers to design and produce custom-made RNA probes of a desired sequence without the need for subcloning, improving the feasibility of generating RNA probes for multiple, discrete target sequences. At high concentrations, the PCR-derived probes produced stronger signals and less background than equivalent plasmid-derived probes, suggesting that his technique may lead to general improvement of in situ hybridization.


  Acknowledgments

Supported by the Swiss National Science Foundation (grant 32-43654)

Received for publication June 13, 1996; accepted December 5, 1996.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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