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Annealing of 18-mer Primer to Spiiced .Sequence of mRNA mid Evlension by rTor rTtli Enzyme

Fig. 3. Overview of multiple splicing system and design for exon-specific primers.

method of getting rid of RNases. In our opinion, one needs to use both auto-claving, as well as DEPC treatment to destroy RNases (18,21). Many researchers believe that autoclaving is not effective at eliminating RNase in solution, because the RNases simply renature as the solution cools; this is particularly not true. Autoclaving destroys the majority of the RNase contamination. However, if your samples contain a large amount of RNases, than both DEPC and autoclaving would be required (21,22).

Generally, autoclaving will indeed inactivate a substantial amount of RNase A. For example, in some reports (15,23) various concentrations of RNase A were added to PBS and autoclaved. Aliquots of each solution were mixed with a 304-base 32P-labeled RNA probe and incubated at 37°C for 1 h, followed by electrophoresis and exposure to film. Without autoclaving, the probe begins to degrade at an RNase concentration of 100 pg/mL. Autoclaving inactivated enough of the RNase A to protect the probe from degradation up to a concentration of 1 ^g/mL. Therefore, only a portion of the RNase was inactivated by autoclaving; otherwise, the RNA probe would remain intact at any RNase concentration. Autoclaving alone may be sufficient to eliminate enough RNases for some applications. However, because neither the extent of RNase con tamination nor at what RNase concentration the assay is sensitive is known, DEPC should be used to make sure that all of RNases are destroyed.

One should keep in mind that autoclaving does inactivate DEPC by causing hydrolysis of DEPC. CO2 and ethanol are released as reaction byproducts. DEPC has a half-life of approx 30 min in water, and at a DEPC concentration of 0.1%, solutions autoclaved for 15 min/L can be assumed to be DEPC-free. After autoclaving the DEPC containing solution, a faint ethanol smell may linger after autoclaving, but more commonly a sweet, fruity smell is observed, which is caused by the ethanol byproduct combining with trace carboxylic acid contaminates and forming volatile esters. The smell is not the result of trace DEPC remaining in the solution.

Of note, DEPC does not inactivate RNases in Tris. Tris contains an amino-group which reacts with DEPC and makes it unavailable to inactivate RNase (19). Tris and HEPES do indeed make DEPC unavailable to inactivate RNase at a DEPC concentration of 0.1% (recommended by most protocols). However, 1% DEPC is sufficient to overcome this effect. However, one has to be concious, as high levels of residual DEPC or DEPC byproducts in a solution can inhibit some enzymatic reactions or chemically alter (carboxymethylate) RNA. It has been documented that DEPC byproducts in RNA samples can inhibit in vitro translation reactions (15-19). It would be difficult to predict the different interactions of DEPC with various molecular biology reagents that are being used in a RT-reaction in situ. The most cautious approach for making RNase-free solutions would be to mix molecular biology-grade powdered reagents in DEPC-treated water. Alternatively, many premade nuclease-free solutions can be purchased from many different commercial sources.

4.7. RT Reaction

Next, one wishes to make cDNA of the targeted RNA sequence so that the signal can be amplified. One should follow the manufacturer's suggestions to prepare the cocktail. After preparing the RT-PCR solution, one should follow the protocol described in Subheading 3.4.2.

4.8. RT-Primers and Target Sequences

In our laboratory, we simply use antisense downstream primers for our gene of interest, as we already know the sequence of most genes we study. However, one can alternatively use oligo (dT) primers to first convert all mRNA populations into cDNA, and then perform the in situ amplification for a specific cDNA. This technique may be useful when performing amplification of several different gene transcripts at the same time in a single cell. For example, if one is attempting to detect various cytokine expressions, one can use an oligo (dT) primer to reverse transcribe all of the mRNA copies in a cell or tissue section.

Then, one can amplify more than one type of cytokine and detect the various types with different colored probes that develop into different colors.

In all RT reactions, it is advantageous to reverse-transcribe only relatively small fragments of mRNA (<1500 bp). Larger fragments may not completely reverse-transcribe because of the presence of secondary structures. Furthermore, the RT enzymes—AMVRT and MMLVRT, at least—are not very efficient in transcribing large mRNA fragments. However, there are several second-generation RT enzymes now available that are more efficient then their predecessors.

4.9. Thermal Cyclers

Various technologies of thermocyclers will work in this application; however, some instruments work much better than others. We use dedicated slide thermocyclers that are specifically designed to hold 16 or 32 slides. We understand that other labs have used stirred-air, oven-type thermocyclers quite successfully; however, we have also heard that there are sometimes problems with the cracking of glass slides during cycling. Thermocyclers dedicated to glass slides are now available from several vendors, including Barnstead Thermolyne of Iowa, Coy Corporation of Minnesota, Hybaid of England, Perkin Elmer of California, and MJ Research of Massachusetts. Our laboratory has used an MJ Research PTC-100-16MS, DNA-Engine Twin-Tower 16 X 2 quite successfully. Recently, this company has combined the slide and tubes into a single block, allowing the simultaneous confirmation of in situ amplification in a tube. Furthermore, there are newer designs of a thermal cycler that incorporate humidification chambers as well temperature gradient to optimize the annealing temperatures for PCR. The gradient thermocyclers are especially useful in the optimization of annealing steps, reverse transcription, and hybridization steps.

We suggest that you follow the manufacturer's instructions on the use of your own thermocycler, bearing in mind the following points: (1) Glass does not easily make good thermal contact with the surface on which it rests. Therefore, a weight to press down the slides and/or a thin layer of mineral oil to fill in the interstices will help thermal conduction. If using mineral oil, make certain that the oil is well smeared over the glass surface so that the slide is not merely floating on air bubbles beneath it; (2) the top surfaces of slides lose heat quite rapidly through radiation and convection; therefore, use a thermocycler which envelopes the slide in an enclosed chamber (as in some dedicated instruments), or insulate the tops of the slides in some manner. Insulation is particularly critical when using a weight on top of the slides, for the weight can serve as an unwanted heat sink if it is in direct contact with the slides; and (3) good thermal uniformity is imperative for good results—poor uniformity or irregular thermal change can result in cracked slides, uneven amplification, or completely failed reactions. If adapting a thermocycler that normally holds plastic tubes, use a layer of aluminum foil to spread out the heat.

4.10. Direct Incorporation of Nonradioactive Labeled Nucleotides

Several non-radioactive labeled nucleotides are available from various sources (i.e. dCTP-Biotin, digoxin II-dUTP, etc.). These nucleotides can be used to directly label amplification products; then, the proper secondary agents and chromogens can be used to detect the directly labeled in situ amplification products. However, the greatest specificity is only achieved by conducting amplification followed by subsequent in situ hybridization. In the direct labeling protocols, nonspecific incorporation can be significant, and even if this incorporation is minor, it still leads to false-positive signals similar to nonspecific bands in gel electrophoresis following solution-based DNA- or RT-amplification. In case of a solution-based PCR, one generally does not notice the nonspecific amplification bands if it is less than 0.2 pg. However, in the case of in situ PCR, in which one is working at the single-cell level, a minutely amplified signal can be observed easily under a high magnification microscope. Therefore, we strongly discourage the direct incorporation of labeled nucle-otides as part of an in situ amplification protocol.

4.11. Multiple Signals, Multiple Labels in Individual Cells

DNA, mRNA, and protein can all be detected simultaneously in individual cells (12,13). One can label proteins by rhodamine-labeled antibodies. Then, one can perform both RNA and DNA in situ amplification in the cells. If one is using primers for spliced mRNA and if these primers are not going to bind any sequences in DNA, then both DNA and RT amplification can be conducted simultaneously. Of course one still needs to perform the RT-step, but this time without pre-DNase treatment. Subsequently, products can be labeled with different kinds of probes, resulting in different colors of signal. For example, proteins can have a rhodamine-labeled probe, mRNA can show a fluorescein isothiocyanate signal (fluorescein isothiocyanate-conjugated probe, more than 20 different fluorochromes are available) and DNA can be labeled with a biotin-peroxidase probe or a fluorochrome with different color emission. Each will show a different signal within an individual cell (12,13).

5. Hybridization

The in situ hybridization (ISH) technique has been successfully applied in both the research and clinical settings. However, one single, easy-to-use universal procedure has not been developed. Therefore, the specific needs of the diagnostic or research goals must be considered in choosing a suitable protocol. We will leave this up to each of the investigators to determine their opti-

mal protocol. Following are some general basic information that needs to be kept in mind.

The most successful procedure would be where an investigator has optimized the ISH of a specific probe with the target genes (in this case the amplicons in the cells of interest) while minimizing background signals (reviewed in [15]). It is the critical factor for ISH sensitivity. Background signals arise primarily from nonspecific retention of a probe in tissue sections (resulting from electrostatic interactions between probe and tissue macromolecules) and entrapment of probes in the spherical lattice of the tissue section (24-30). Several chemically functional groups in proteins (i.e., carboxylate and amine groups) may be responsible for this nonspecific binding. There are several protocols to minimize the background signals by treating tissue slides with acetic anhydride and tri-ethanolamine (27). Acetylation of amine groups by acetic anhydride, routinely used in ISH protocols, maybe important in reducing backgrounds for probes larger than 2.0 kb (29).

Another way to decrease nonspecific probe binding is to saturate the binding sites on proteins by incubating tissues with prehybridization solution, which typically includes Ficoll, bovine serum albumin, polyvinyl pyrrolidone, and nucleic acids. These reagents also are present in hybridization buffers to compete with the nonspecific binding of probes to tissue. However, this is not a fool-proof method, and it does not completely prevent background signals. Nuclease treatment after hybridization is still necessary for reducing this nonspecific signal (nuclease treatment degrades unhybridized, single-stranded probes). Some investigators have found that without RNase treatment, the background with [33P]-labeled RNA probes was so high that specific hybridization signals was not discernable. Even high stringency washing did not remove this background. Generally, RNA probes tend to exhibit high levels of nonspecific binding, so RNase treatment must be applied in ISH studies when RNA probes are used (30).

Antisense RNA probes are used widely for ISH because they have been demonstrated to be more specific and sensitive than cDNA probes or oligoprobes (15,24). Both isotopically and nonisotopically labeled probes have been used successfully for ISH. 33P and 35S frequently are used isotopes to label probes. 35S-labeled RNA probes usually give higher backgrounds, so when 35S-labeled probes are used, dithiothreitol should be added to all solutions used in prehybridization, hybridization, and posthybridization washes. If one is going to use radioisotope labeled probes, then we recommend using 33P-labeled probes for ISH studies because they result in lower background and higher resolution as compared with 35S-labeled RNA probes.

Nonisotopic labeled probes (i.e., biotin, or chemiluminescent labeled) are the most frequently used for ISH studies. The sentitivity of these probes may be significantly lower than the radioisotopic probes but for post-in situ PCR, they are the ideal probes to be used.

Although there are different recipes for making hybridization buffers, the inclusion of dextran sulfate in the hybridization solution increases probe binding to target mRNA, that is, including 10% dextran sulfate enhances ISH signal several fold. However, too much dextran sulfate in the hybridization buffer will induce high background, which is difficult to remove in posthybridization washes. Various methods to be used for different kinds of applications have been described in detail in several of our previous publication (15,20,31).

6. Controls

The validity of in situ amplification-hybridization should be examined in every run. Attention here is especially necessary in laboratories first using the technique because occasional technical pitfalls lie on the path to mastery. In an experienced laboratory, it is still necessary to continuously validate the procedure and to confirm the efficiency of amplification. To do this, we routinely run two or three sets of experiments in multiwelled slides simultaneously, for we must not only validate amplification, but we must also confirm the subsequent hybridization/detection steps.

In our laboratory, we frequently work with infectious agents. A common validation procedure we conduct is to mix infected cells with uninfected cells in known ratios (i.e., 1:10, 1:100, etc.), and then confirm that the results are appropriately proportionate. To examine the efficiency of amplification, one can use a cell line, which carries a single copy, or two copies of the gene of interest (1,13,32-35), and then look to see that proper amplification and hybridization has occurred.

In all amplification procedures, we use one slide as a control for nonspecific binding of the probe. Here we hybridize the amplified cells with an unrelated probe. We also use human leukocyte antigen-DQ and P-actin probes and primers with human peripheral blood mononuclear cells and other tissue sections as positive controls to check various parameters of our system.

In case one is using tissue sections, a cell suspension lacking the gene of interest can be used as a control. These cells can be added on top of the tissue section and then retrieved after the amplification procedure. The cell suspension can then be analyzed with the specific probe to see if the signal from the tissue leaked out and entered the cells floating above.

We suggest that researchers carefully design and use appropriate positive and negative controls for their specific experiments. In the case of RT-in situ amplification, one can use P-actin, y-globulin, human leukocyte antigen-DQa, and other endogenous-abundant RNAs as the positive markers. Of course, one should always have an RT-negative control for RT in situ amplification, as well as DNase and non-DNase controls. Controls without polymerase plus primers and without primers should always be included.

References

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2. Lattime, E. C., Mastrangelo, M. J., Bagasra, O., Li, W., and Berd, D. (1995) Expression of cytokine mRNA in human melanoma tissue. Cancer Immunol. Immunother. 41, 151-156.

3. Pestaner, J. P., Bibbo, M., Bobroski, L., Seshamma, T., and Bagasra, O. (1994) Potential of in situ polymerase chain reaction in diagnostic cytology. Acta Cytol. 38, 676.

4. Patterson, B., Till, M., Otto, P., et al. (1993) Detection of HIV- I DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry. Science 260, 976-979.

5. Maggioncalda, J., Mehta, A., Bagasra, O., Fraser, N. W., and Block, T. M. (1996) A herpes simplex virus mutant with a deletion immediately upstream of the lat locus establishes latency and reactivates from latently infected mice with normal kinetics. J. NeuroVirol. 2, 268-278.

6. Sullivan, D. E., Bobroski, L. E., Bagasra, O., and Finney, M. (1997) Self-seal reagent: evporation control for molecular histology procedure without chambers, clips or fingernail polish. BioTechniques 23, 320-325.

7. Winslow, B. J., Pomerantz, R. J., Bagasra, O., and Trono, D. (1993) HIV-1 Latency due to the site of proviral integration. Virology 196, 849-854.

8. Bobroski, L., Bagasra, A. U., Patel, D., et al. (1999) Localization of Human Herpes Virus Type 8 (HHV-8) in the Kaposi's Sarcoma tissues and the semen specimens of HIV-1 infected and uninfected individuals by utilizing in situ polymerase chain reaction. J. Reprod. Immunol. 41, 149-160.

9. Ewida, A. S., Raphael, S., and Bagasra, O. (2002) The presence of IL-2 and IL-10 cytokines in the skin lesions of Blau Syndrome. Appl. Immunochem. Mol. Morphol. 10, 171-177.

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14. Bobroski, L., Bagasra, A. U., Patel, D., Saikumari, P., Memoli, M., Wood, C., Sosa, et al. (1999) Mechanism of vertical transmission of HIV-1: role of intervillous space. Appl. Immunochem. Mol. Morphol. 7, 271-279.

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29. Lynn, D. A., Angerer, L. M., Bruskin, A. M., Klein, W. H., and Angerer, R. C. (1983) Localization of a family of mRNAs in a single cell type and its precursors in sea urchin embryos. Proc. Natl. Acad. Sci. USA 80, 2656-2600.

30. Valentino, K. L., Eberwine, J. H., and Barchas, J. D. (1987) In Situ Hybridization: Application to Neurobiology. Oxford University Press, New York, pp. 179-196.

31. Bagasra, O., Hauptman, S. P., Lischner, H. W., Sachs, M., and Pomerantz, R. J. (1992) Detection of HIV-1 provirus in mononuclear cells by in situ PCR. N. Engl. J. Med. 326, 1385-1391.

32. Embretson, J., Zupancic, M., Beneke, J., et al. (1993) Analysis of human immunodeficiency virus-infected tissues by amplification and in situ hybridization reveals latent and permissive infections at single-cell resolution. Proc. Natl. Acad. Sci. USA 90, 357-436.

33. Embretson, J., Zupancic, M., Ribas, J. L., et al. (1993) Massive covert infection of helper T lymphocytes and macrophages by HIV during the incubation period of AIDS. Nature 62, 359-362.

34. Harrington, W. Jr., Bagasra, O., Sosa, C. E., et al. (1996) Human Herpes Virus 8 (HHV-8) DNA sequences in cell free plasm and mononuclear cells in AIDS and non-AIDS patients. J. Infect. Diseases 174, 1101-1105.

35. Nuovo, G. J. (1994) PCR In Situ Hybridization Protocols and Applications (2nd ed). Raven Press, New York.

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