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RT In Situ PCR 451 coverslips anchored with nail polish, and polyethylene bags, cut to size and held in place with nail polish (5,17). It is important to remove ALL visible air bubbles because these will be expanded as a result of the cycling process, which can result in some parts of sections being inaccessible to the PCR reagents, resulting in false negatives. Different means of covering the cell spots will result in different volumes of reagent required for each step, and they should be adjusted accordingly to save costs. 5. Protease digestion reduces the protein cross-links that form during the fixation step, thus allowing reagents to enter the cell. We currently use pepsin (5000 U/mL) at 37°C. The duration of pepsin digestion depends on the type of fixative used and the extent of fixation, with mast cell, macrophage, and lymphocyte cell lines taking ~45 min of digestion, whereas other cells such as in vivo derived mast cells requiring much less digestion (~15 min). However, other temperatures (22°C) and enzymes (trypsin, proteinase K) have been used by various groups (7). Both these enzymes can give excellent results as well. We have settled on pepsin as our enzyme of choice, as it works at low pH (3.0), and is thus easily inactivated by a wash in water (~pH 7.5). 6. As a first step in the process of optimizing the digestion time and to become familiar with the methodology, we use a shortened protocol (5). This involves using a single slide, with each individual spot being subjected to a different digestion time (e.g., 20 min, 40 and 60 min). After washing off the protease, the slide is then subjected to PCR and development, with no DNase or RT steps. By looking at the nuclear staining obtained with the different times, the investigator will be able to pick the time that gives >90% of the nuclei positive but maintains the cellular architecture. This optimal time can then be used for complete RT in situ PCR studies on this same batch of fixed cells. Some investigators have noted that they use >50% of the nuclei positive as an indication of optimal digestion (4,5). We generally see >90% positive nuclei and have chosen this as our optimal indicator of digestion. 7. Recommended primer lengths are between 20 to 30 nucleotides. The PCR product should be at least 300 bp in length to assure that the amplicons remain inside the cell. The RT solution should contain an antisense primer capable of initiating first strand cDNA production. Oligo-dT can be used, although we have found that a gene specific primer works best. 8. Numerous RT enzymes are commercially available. We have used both Maloney Murine Leukemia Virus (MMLV) and Avian Myeloblastosis Virus (AMV) RT enzymes with identical results. We do our RT steps at 37°C, and vary the time of incubation from 1 to 3 h, depending on the signal seen with a specific cell type. 9. The critical parameter in the composition of the PCR solution is the MgCl 2 concentration. We found that Mg 2+ concentration in the range of 3.0 to 5.0 mM works best, which is about four times higher than that used in solution-based PCR. This necessary increase in Mg 2+ concentration is thought to be caused by binding of Mg 2+ to the glass slides (5,7). 10. If no product is detected with the cycling program given, then the program will have to be optimized. Take into account the T m of the primers being used, and vary the number of cycles. In general terms, we begin with an annealing temperature that is 5°C below that of the primer Tm. Furthermore, depending on the abundance of the mRNA being amplified, upwards of 35 cycles may be required to obtain a detectable signal. 11. Transposed onto the technique of in situ RT-PCR is the necessity to detect the amplified product using immunohistochemical techniques. This introduces numerous other variables into the protocol. We have found that antibody concentrations of 0.75 to 3.75 µg/mL provide optimal signal with little background staining. Acknowledgments The authors recognize Dr. Osamu Nohara for his contribution to the development of our understanding of this procedure.
452 Gilchrist and Befus References 1. Mullis, K. B. and Faloona, F. A. (1987) Specific synthesis of DNA in vitro via a polymerasecatalysed chain reaction. Methods Enzymol. 155, 335–350. 2. Veres, G., Gibbs, R. A., Scherer, S. E., and Caskey, C. T. (1987) The molecular basis of the sparse fur mouse mutation. Science 237, 415– 417. 3. Long, A. A. (1998) In situ polymerase chain reaction: foundation of the technology and today’s options. Eur. J. Histochem. 42, 101–109. 4. Nuovo, G. J., Forde, A., MacConnell, P., and Fahrenwald, R. (1993) In situ detection of PCR-amplified HIV-1 nucleic acids and tumor necrosis factor cDNA in cervical tissues. Am. J. Pathol. 143, 40– 48. 5. Nuovo, G. J. (1992) PCR in situ Hybridization: Protocols and Applications. Raven Press, New York. 6. Chen, R. H. and Fuggle, S. V. (1993) In situ cDNA polymerase chain reaction. Am. J. Pathol. 143, 1527–1534. 7. Teo, I. A. and Shaunak, S. (1995) Polymerase chain reaction in situ: An appraisal of an emerging technique. Histochem. J. 27, 647–659. 8. Nohara, O., Gilchrist, M., Dery, R. E., Stenton, G. R., Hirji, N. S., and Befus, A. D. (1999) Reverse transcriptase in situ polymerase chain reaction for gene expression in rat mast cells and macrophages. J. Immunol. Methods 226, 147–158. 9. Gilchrist, M., MacDonald, A. J., Neverova, I., Ritchie, B., and Befus, A. D. (1997) Optimization of the isolation and effective use of mRNA from rat mast cells. J. Immunol. Methods 201, 207–214. 10. Chiu, K. P., Cohen, S. H., Morris, D. W., and Jordan, G. W. (1992) Intracellular amplification of proviral DNA in tissue sections using the polymerase chain reaction. J. Histochem. Cytochem. 40, 333–341. 11. Patterson, B. K., Till, M., Otto, P., Goolsby, C., Furtado, M. R., McBride, L. J., et al. (1993) Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry. Science 230, 1350–1354. 12. Hagimoto, N., Kuwano, K., Nomoto, Y., Kunitake, R., and Hara, N. (1997) Apoptosis and expression of Fas/Fas ligand mRNA in bleomycin-induced pulmonary fibrosis in mice. Am. J. Respir. Cell Mol. <strong>Bio</strong>l. 16, 91–101. 13. Peters, J., Krams, M., Wacker, H. H., Carstens, A., Weisner, D., Hamann, K., et al. (1997) Detection of rare RNA sequences by single-enzyme in situ reverse transcription polymerase chain reaction: High-resolution analysis of interleukin-6 mRNA in paraffin sections of lymph nodes. Am. J. Pathol. 150, 469– 476. 14. O’Leary, J. J. (1994) Importance of fixation procedures on DNA template and its suitability for solution phase PCR and PCR in situ hybridization. Histochem. J. 26, 337–346. 15. Long, A. A., Komminoth, P., and Wolf, H. J. (1993) Comparison of indirect and direct in situ polymerase chain reaction in cell preparations and tissue sections. Histochemistry 99, 151–162. 16. Teo, I. A. and Shaunak, S. (1995) PCR in situ: aspects which reduce amplification and generate false-positive results. Histochem. J. 27, 660–669. 17. Patel, V. G., Shum-Siu, A., Heniford, B. W., Weiman, T. J., and Hendler, F. J. (1994) Detection of epidermal growth factor receptor mRNA in tissue sections from biopsy specimens using in situ polymerase chain reaction. Am. J. Pathol. 144, 7–14.
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Xin Wang and W. Scott Young III 105
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63. Primed In Situ Nucleic Acid Lab
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Table 1 Oligonucleotides Descriptio
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504 Ravassard et al. 9. ddH 2 O. 10
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506 Ravassard et al. 3.2.2. RNA Mix
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512 Pont-Kingdon Fig. 1. Constructi
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530 Burke and Barik purified mutant
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532 Burke and Barik
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