In Situ Hybridization MADELEINE DUVIC, MD
A
lthough several molecular biology techniques can be used to measure mRNA, only in situ hybridization (or in situ transcription) permit specific localization of DNA or an mRNA species within a tissue section or cell preparation. With appropriate fixation, mRNAs can be preserved and detected in tissue sections by using DNA or RNA probes labeled with radioactive or chemically modified nucleotides. In situ hybridization has been widely used in molecular and developmental biology to understand gene regulation in tissues and to localize the position of cloned genes on chromosomes, With the introduction of non-isotopic labeling techniques and commercial kits, in situ can be useful for detecting viral RNA or DNA or other genes of interest (like cancer-related oncogenes) in tissue specimens. The development of in situ hybridization is to molecular biology as immunofluorescence and immunoperoxidase staining have been to protein biochemistry, enabling localization and temporal relation of specific mRNAs .
Background In order to perform in situ hybridization, one must first isolate a probe for the gene of interest. Most commonly, this has been a cDNA complementary to the messenger RNA. cDNA isolation and cloning is a time-consuming and sometimes difficult project, which precedes one’s ability to do in situ hybridization, unless the cDNA has already been cloned. Hybridization of one strand of nucleic acid to another, whether RNA or DNA, depends on naturally occurring From the Department of Dermatology and internal Medicine, Dermatology Section, The University of Texas Medical School, M.D. Anderson Cancer Center, Houston, Texas. Address correspondence to: Madeleine Duvic, M.D., Department of Dermatology and Internal Medicine, M.D. Anderson Cancer Center, Houston, TX 77030.
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complementary base pairing between cytosine and guanine and between adenosine and either thymidine (DNA) or uracil (RNA). Heating cDNA melts the complementary strands, creating two single-stranded DNAs (called probes) that reanneal with DNA-complementary sequence to give a signal, Likewise, one can detect singlestranded RNA by the appropriate anti-sense complementary DNA single strand or by producing an anti-sense complementary RNA (riboprobe). In the standard Southern’ and Northern hybridizatier? techniques, DNA or RNA is first purified from tissue, separated by fragment size using electrophoresis, and transferred to a membrane for liquid hybridization with labeled probes. Both Southern and Northern hybridizations require the isolation of large (microgram) quantities of nucleic acids. In the isolation procedure, low copy number genes or RNAs are diluted, sometimes below the level of detection for the blotting techniques. By measuring nucleic acids in situ one may detect as little as one copy of a nucleic acid sequence. In situ hybridization techniques were first adapted to preparations of cells and chromosomes3T4 and used to detect amplified genes. 3,5,6Improvements in tissue fixation and labeling have made it possible to detect single copies of viral genomes in cells3 and single genes on chromosomes.‘,* Thus, many research applications of in situ hybridization include developmental and tumor biology, virology and microbiology, gene mapping and expression. Clinically, it has applications for improved cytogenetics and detection of genetic disease, cancer, and infectious organisms.9
Technical Aspects of In Situ Hybridization The basic method for in situ hybridization is shown in Figure 1. Included are a number of steps that differ slightly depending on the kind of probe used, how it will be labeled, and whether DNA or mRNA is to be detected.
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preparations are dehydrated through alcohols and embedded in paraffin. Skin sections should be cut at 4 - 5 ,um thickness or less to avoid high background and trapping of probe. In some cases, to facilitate probe penetration, the tissue sections may be digested with a limited proteinase K digestion step or with HCl, heat, or detergent.3*10-14 Protease treatment may be more critical with the use of a glutaraldehyde-based fixative.3 The goal of tissue fixation is to optimally preserve the morphology of the tissues while stabilizing the mRNA and making it most accessible to the hybridization probe.
Outline of the in situ hybridization procedure.
C. Probe Preparation Excellent overviews of the procedure and its permutations include the following review articles.3*10-*2
A. Slide Preparation Tissue specimens are subjected to repeated washings and will often become detached unless special procedures are taken to make slides used more adherent. Soaking tissue sections in Elmer’s glue diluted in the waterbath prior to slide application works well for paraffin-embedded tissue used for DNA hybridization. However, for hybridization to mRNA, all contaminating RNase enzymes must be destroyed on the slides. We use the procedure adapted from Cox et a113-15 in which slides are washed in 10% Extran 300 overnight, washed exhaustively, and baked at 160°C. The slides are then coated with 2% 3-aminopropyltriethyloxysilane (Sigma) in acetone followed by RNase free water and dried at 42”C.15
B. Tissue Preparation
and Preservation
For detection of DNA (such as papilloma virus genome), routine fixation with buffered formalin followed by routine paraffin embedding and fixation is adequate.16*” As mRNAs are extremely labile and degraded by ubiquitous enzymes (RNases), preservation of RNA is critical. All equipment and glassware must be specially treated or sterile plastic used, and gloves must be used. Tissue can be snap frozen in liquid nitrogen and later cryosectioned, then fixed on the slide in paraformaldehyde according to the method of Harper.16 Alternatively, immediate fixation in 4% freshly made paraformaldehyde, which cross-links the mRNA just enough to allow excellent probe penetration with retention of the tissue morphology,14 can be used. Other fixatives that have been used for in situ hybridization include glutaraldehyde, formaldehyde, ethanol, or methanol.3J0-14 Following fixation, the tissue or cytospin
and Labeling
DNA Probes Single-stranded cDNA probes can be synthesized using phage Ml3 vectors or from viral RNA templates using reverse transcriptase. Cloned cDNA probes can be labeled by random primer method19 and melted to create single-stranded probes. In the latter case, there is competition between the annealing of the two complementary probe strands with the nucleic acid to be detected by in situ method.
RNA Probes A major improvement has been the development of plasmid vectors (pGEM, Promega, and others) containing Sp6 and T7 viral RNA polymerase promoters. These promoters flank a cassette of multiple restriction enzymes, which can be used to insert a cDNA of interest. Using the two promoters, single-stranded riboprobes are transcribed in opposite directions to generate sense and antisense strands that serve as negative and positive probes for mRNA molecules.15 These riboprobes are conveniently labeled during the transcription procedure with radioactive or biotinylated uridine triphosphate (UTP). Transcription and cloning kits are now available commercially. There are several major advantages to the riboprobes for in situ hybridization. Large microgram quantities of probe can be labeled to high specific activity. Following hybridization, unbound single-stranded probe can be removed by RNase treatment. RNase treatment plus the higher thermal stability of RNA-RNA duplexes compared to DNA-RNA hybrids allow higher washing temperatures, and reduce the non-specific background hybridization. For optimal penetration of riboprobes, reduction to 100 - 200 nucleotides is suggested in some protocols. This is achieved by limited alkaline lysis.13J4
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Control Probes Proper in situ hybridization technique demands the inclusion of control probes to demonstrate that binding is specific and not due to vector sequence or to non-specific hybridization. Riboprobes transcribedinoppositeorientations serve this purpose for detection of mRNA by the antisense strand. For detection of viral nucleic acids, it is important to include specimens known to be positive by other detection methods and to use heterologous probes from viruses not expected to be encountered. In some cases, it may be necessary to show that mRNA in the sample has not been degraded using a probe for a constituitively or abundantly expressed mRNA.
D. Hybridization
Conditions
During the hybridization step, the probe is allowed to anneal to the DNA or RNA within the specimen. The temperature, which is influenced by type, length, and sequence of probe used, is a critical parameter. Optimum temperature has been suggested as 25 “C below the melting temperature of the hybrids formed.14 The use of formamide in the reaction allows hybridization at lower temperatures but formamide is light sensitive. Hybridization is best performed under a coverslip in a humidified air chamber. Special hybridization “Probe-On” slides have been suggested by some to give superior results.*O To avoid high background, the probe concentration should be the lowest possible to still saturate the target mRNAs in a reasonable amount of time.3 Generally, a hybridization time of under 4 hours is recommended to keep background low. For a discussion of the reaction kinetics, the reader is referred to several reviews.3J0-14 The probe, if initially doublestranded, competes with its other strand for annealing to RNAs. Following the hybridization, excess unbound probe is removed by a series of stringency washes using excess volume of sodium citrate buffer and sodium dodecylsulfate. If riboprobes were used, unbound probes are digested with RNase during the wash procedure.i3,14
E. Detection Systems Radioactive Probes These have been most commonly used for in situ hybridization. Following washing to remove unbound probe, the slides are dipped in Kodak NTB2 emulsion, air dried, and autoradiographed at 4”C-70°C for a period of several days to weeks. t* The slides are then developed and fixed as by standard photography and analyzed by light and dark field microscopy. The number of grains per cell minus the background can be quantitated using a digitizer. Because of the problems interpreting a positive signal from background hybridization, we have used con-
Figure 2. In situ hybridization using %-UTP-labeled riboprobe to detect cells infected with the human immunodeficiency virus (HIV). (top): Bright field. (middle): Dark field. (bottom): Same field analyzed using confocal microscopy detectionz’Jz shows marked reduction in non-specific background.
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focal microscopy with digital image processing to determine specific hybridization signals.21*22 Figure 2 shows cells hybridizing with HIV riboprobe as detected by light microscopy (Figure 2A) and dark field (2B). The use of the confocal microscope results in elimination of the nonspecific background hybridization (Figure 2C). The choice of radioisotopes for in situ hybridization includes tritium 3H-, 32P-, and 35S-substituted nucleotide triphosphates. 3H is the most stable and gives the best localization with the lowest background signal but may take several months of autoradiography to give a detectable signal. 32P gives a strong sign al in the shortest period of time, but gives high background and poor localization. Most investigators use 35S as a compromise between the other two isotopes because it gives high specific activity and reasonable background.
Non-lsotopic Detection Alternative detection methods use cytochemical detection reactions, especially biotinylation with avidin-labeled detection using enzymatic or fluorescent molecules as reporters. Horseradish peroxidase and alkaline phosphatase are commonly used for enzymatic detection. Although these detection systems may be as sensitive as radionucleotides for detecting low-copy-number RNAs,‘O*” it is difficult, if not impossible, to quantitate amounts of mRNA detected at the present time. The advantage of cytochemical detection is the short period of time required for the analysis, compared to autoradiography, and the stability of probes, making them useful for clinically based diagnostics.
Applications of In Situ Hybridization Development
Biology
The advantage of in situ hybridization as a technique is its ability to localize low copy numbers of a specific mRNA or DNA molecule in a cell or tissue. In situ can be used to understand the timing and patterns of gene expression in embryogenesis, tissue differentiation, and disease. This is perhaps its most important research application at the current time. Cancer Biology In situ hybridization can be used to detect the breakpoint in tumor-related translocations, as well as to detect expression of cancer-related oncogenes. These can also be detected using Southern blotting and the more sensitive Polymerase Chain Reaction (PCR).
Gene Mapping The cloning of cDNAs for specific genes has been followed by an intense effort to map their location on chro-
mosomes. This can be best accomplished by a combination approach where the gene is localized to a specific chromosome, using somatic cell panels, and then to a specific chromosome band, using in situ hybridization.25,26 Somatic cell hybrid panels are generated by fusing animal (usually Chinese Hamster ovary cells) with human cells and selecting hybrid cells that contain one or more human chromosomes, or parts of chromosomes.25 DNA from each hybrid cell line is prepared and hybridized to labeled cDNA probes by standard Southern hybridization.’ By analyzing which lines hybridize to the cDNA probe, discordancy to the human chromosomes can be determined.25,26 When fractions of human chromosomes are found within hybrids, more specific localization to long or short arms can be made. Once the chromosome localization is determined, in situ hybridization can be a powerful tool to find the most probable gene location within one or more bands9r2’ For this procedure, white blood cells are stimulated to divide with phytohemaglutanin (PHA) and metaphase chromosomes are isolated and banded. In situ hybridization with 3H-labeled cDNA probes is then performed and analyzed by microscopy following autoradiography. The number of grains hybridizing to each chromosome are plotted from a number of cell preparations.27 Numerous genes have been mapped using this technique.27-29
Cytogenetics A technical advance in in situ hybridization has recently been developed that will enable researchers to determine the orientation of genes along one chromosome fragment.30 By using biotinylated cDNA probes detected by immunofluorescence dyes of different colors, it has been possible to orient several genetic markers along a stretch of a chromosome and therefore to determine probable gene order. 3oBecause in situ hybridization using immunofluorescent probes is so quick, and can be accomplished in 1 - 2 days, it may also become a very important cytogenetic tool with clinical application for determining chromosomal aneuploidy, such as Down’s Syndrome, in specimens collected by chorionic villus sampling or by amniocentesis.30,31 Detection of genetic disease may be accomplished in diseases in which large pieces of DNA are deleted, such as Duchenne’s muscular dystrophy and X-linked ichthyosis. In Situ Hybridization
Can Detect Viral Nucleic Acids
One of the earliest clinical applications of in situ hybridization was to detect human viruses in cells and tissue specimens using biotinylated32 or radioactive DNA probes.3 Human papilloma viruses (HPV) are DNA viruses that have been implicated in the etiology of squa-
Clinics in Dermatology 1992;9:229-235 mous cell carcinomas, especially of the anogenital tract. Earliest studies for detection of viral DNA were dot-blot hybridizations using extracted DNA. However, dot-blots only tell whether a nucleic acid is present or absent and do not give information regarding which cells are infected.33 As HPV types 16 and 18 have been implicated as more carcinogenic, determination of the specific HPV type has been of clinical interest in studying specimens from cervical and anogenital carcinomas, and from condylomata by In addition to tissue speciin situ hybridization. 14~15,34-36 mens, cervical cells from Pap smears have also been studied for HPV DNA sequences and have shown a high prevalence of HPV 16 and 18 sequences in severe dysplasia and carcinoma.35,37 A viral detection kit for Pap smear in situ hybridization is commercially available. In situ hybridization has also been used to demonstrate the presence of HPV in oral carcinomas3* and recently in other squamous cell carcinomas of the head and neck39 and in keratoacanthomas.40 Typing of HPV by in situ hybridization, at least for the handful of more virulent strains, is a feasible, clinical application of the technique using biotinylated DNA probes in paraffin-embedded specimens or cytology preparations, and is more sensitive than routine Pap smears in detecting HPV. Other DNA viruses have also been detected using in situ hybridization. 41 Abnormal human cervical tumor biopsies were found to contain Herpes simplex type 2 (67%), and adenovirus type 2 (39%) using 3H-labeled DNA probes versus 23% and 17% of normal biopsies.42 However, because tissue specimens may contain bacterial contamination including plasmids, which cross hybridize with the vector used to make probes,43 the appropriate vector control probe should be used and give a low percentage of positive signals.42 The mechanism of how herpes simplex virus remains latent in the ganglia was studied in trigeminal ganglion from autopsies using in situ hybridization.M Positive signals were detected in 67% of subjects, similar to the proportion of seropositive individuals. Interestingly, an immediate early gene (ICPO), which is actively expressed during acute infection, was expressed as an antisense transcript, suggesting that antisense RNA may maintain latency of the virus. 44 The power of in situ hybridization for virology is that it will detect provirus incorporated into the DNA or in the cytoplasm in the absence of viral protein or particle expression. This ability to detect latent virus is already changing our concept of the role of viruses in human diseases. Retroviruses are RNA viruses with the capability of incorporating themselves into host DNA using the enzyme reverse transcriptase. They can remain latent in cells without expressing virus or viral particles for long periods of time, and can be inherited through the germ-
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line-like genes. In situ hybridization as well as PCR can be used to detect latent or low copy-number retrovirus infections.3 Standard Southern hybridization techniques, on the other hand, cannot identify nucleic acid sequences present in amounts less than one copy for every 10 - 100 cells.45 The role of retroviruses in causing human diseases has only recently been appreciated with the discovery of HTLV-I in T-cell lymphoma cells.46,47 HTLV-I has also been associated with tropical spastic paraparesis,‘* and is less well documented in association with systemic lupus erythematosus,49 and mycosis fungoides (cutaneous T cell lymphoma).50 The human immunodeficiency virus HIV-I (formerly HTLV-III), is another recently discovered retrovirus of the lentivirus family that infects and destroys the human immune system, causing AIDS and/or severe neurologic dysfunction. 45 In situ hybridization has been used to study the cell populations infected by HIV, including CD4+ T cells,18,51 monocytes,52 microglial cells,53 megakaryocytes,54 and dermal dendritic cells.*l The pattern of infection of cells with HIV may well explain the manifold clinical manifestations associated with this virus,
Comparison of In Situ Hybridization with the Polymerase Chain Reaction The polymerase chain reaction (PCR) is also being widely used to detect viral nucleic acids in cells and tissue.55 As it is many times more sensitive than in situ hybridization and more rapid to perform, it may turn out to be the more clinically useful detection method. However, the sensitivity of PCR may lead to a high incidence of false positives. Furthermore, PCR does not give information regarding the localization of nucleic acids unless performed on single cells or sperm. PCR is the most sensitive detection method possible because of its capability to amplify one copy of DNA many millions of times. Although PCR can also be used to detect mRNA following production of a first-strand cDNA species, and may be made quantitative with the use of an internal standard, this latter procedure is a multistep process that may not lend itself to a routine laboratory test. Thus, in situ hybridization may continue to have clinical applications where cellular or temporal localization of one or more nucleic acids are desired, or where simultaneous detection of protein and mRNAs are required to be visualized. However, in situ hybridization does not easily lend itself to quantitation, especially using enzymatic or fluorescent detection methods. Because in situ hybridization using conventional radioactive probes is a technique requiring fastidious conditions and weeks to perform, it is unlikely to be widely adapted for clinical use. However,
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with the development of commercially available probes labeled non-isotopically for fluorescent or enzymatic reporter detection, clinical applications of the technique may become much more widely used for screening or detection of medically important mRNAs or cytogenetic abnormalities, and of nucleic acids from infectious agents that would be impossible to detect by other means. Acknowledgments This work was supporfed in part by NIH grants R29AR36546 and ROl-AR39975. The author wishes to thank Elaine Fuchs, Ph.D. and Andrea Sfoler, Ph.D. for their help, and acknowledges the research collaboration of Wanda T. Schroeder, Ph.D., Sfeven Mahoney, M.D., and Wanda O’Brien, B.A. Dr. Schroederwas the recipient ofDermatology Foundation Fellowships from Roche Laboratories and Squibb Pharmaceuticals. Dr. Mahoney was the recipient ofa Dermatology Fellowship from Beecham Laboratories.
References 1. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975;89:50317. 2. Thomas PS. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Nat1 Acad Sci USA 1974;71:5201-05. 3. Haase A, Brahic M, Stowring L, Blum H. Detection of viral nucleic acids by In Situ hybridization. In Methods in Virology, New York Academic Press, Inc., 1984, pp 212- 14. 4. John HA, Birnstiel ML, Jones KW. RNA-DNA hybrids at the cytological level. Nature 1969;223:582-87.
12. Gee CE, Roberts JL. In situ hybridization histochemistry: A technique for the study of gene expression in single cells. DNA 1983;2:157-63. 13. Cox KH, DeLeon DV, Angerer LM, Angerer RC. Detection of mRNAs in sea urchin embryos by in situ hybridization suing asymmetric RNA probes. Dev Bioll984,485-502. 14. Angerer RC, Cox KH, Angerer LM. In situ hybridization to cellular RNAs. In Setlow JK, Holleander A, eds. Genetic Engineering, Vol. 7. New York and London, Plenum Press, 1985, pp 43-65. 15. Stoler A, Kopan R, Duvic M, Fuchs E. Use of monospecific antisera and cRNA probes to localize the major changes in keratin expression during normal and abnormal epidermal differentiation. J Cell Biol 1988;107:427-46. 16. Wells M, Griffiths S, Lewis F, Bird C. Demonstration of human papillomavirus types in paraffin processed tissue from human anogenital lesions by in situ DNA hybridization. J Path01 1987;152:77-82. 17. von Krogh G. Advantage of human papilloma virus typing in the clinical evaluation of genitoanal warts. Experience with the in situ deoxyribonucleic acid hybridization technique applied on paraffin sections. J Am Acad Dermatol 1988;18:495-503. 18. Harper ME, Marselle LM, Gallo RC, Wong-Staal F. Detection of lymphocytes expression human T-lymphotrophic virus type III in lymph nodes and peripheral blood from infected individuals by in situ hybridization. Proc Nat1 Acad Sci USA 1986;83:772-76. 19. Feinberg AI’, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1984;137:266-67. 20. Munjaal RF’, Conneely OM, O’Malley BW. In situ detection of progesterone receptor mRNA in the chicken oviduct using probe-on slides. BioTechniques 1989;7:1104-07.
5. Jones KW. Chromosomal and nuclear location of mouse satellite DNA in individual cells. Nature 1970;225:91215.
21. Mahoney SE, Duvic M, Nickoloff BJ, et al. HIV transcripts identified in HIV-related psoriasis and Kaposi’s sarcoma lesions. J Clin Invest (submitted).
6. Henderson AS, Warburton D, Atwood KC. Location of ribosomal DNA in the human chromosome complement. Proc Nat1 Acad Sci USA 1972;69:3394-98.
22. Shotten DM. Confocal scanning optical microscopy and its applications for biological specimens. J Cell Sci 1989;94:175-206.
7. Tereba A, Lai MMC, Murti KG. Chromosome 1 contains the endogeneous RAV-0 retrovirus sequences in chicken cells. Proc Nat1 Acad Sci USA 1979;76:6486-90.
23. Venezky DL, Angerer IM, Angerer RC. Accumulation of histone repeat transcripts in the urchin egg pronuclease. Cell 1981;24:385-91.
8. Harper ME, Ulrich A, Saunders GF. Localization of the human insulin gene to the distal end of the short arm of chromosome 11. Proc Nat1 Acad Sci USA 1981;78: 4458-60.
24. Berman SA, Bursztajn S, Bowen B, Gilbert W. Localization of an acetylcholine receptor intron to the nuclear membrane. Science 1990;247:212-14.
9. Lichter I’, Ward DC. Is non-isotopic in situ hybridization finally coming of age? Nature 1990;345:93-94. 10. Singer RH, Lawrence JB, Villnave C. Optimization of in situ hybridization using isotopic and non-isotopic detection methods. BioTechniques 1986;4:230-45. 11. Bresser J, Evinger-Hodges MJ. Comparison and optimatization of in situ hybridization procedures yielding rapid, sensitive mRNA detections. Gene Anal Techn 1987;4:89- 104.
25. Thompson LH, Carrano AV, Sato K, et al. Identification of nucleotide-excision repair genes on human chromosomes 2 and 13 by functional complementation in hamster-human hybrids. Somat Cell Molec Genet 1987;13:539-51. 26. Sicilian0 MJ, Carrano AV, Thompson LH. Assignment of a human DNA repair gene associated with sister chromatid exchange to chromosome 19. Mutation Res 1986;174: 303-08. 27. Harper ME, Saunders GF. Localization of single copy DNA
Clinics in Dermatology 7991;9:129-135
sequences on G banded chromosomes by in situ hybridization. Chromosoma 1981;83:431-39. 28. Human Gene Mapping 9. In Klinger HP, ed. Cytogenetics and Cell Genetics. Basel, S. Karger, 1987, p 46. 29. Schroeder WT, Lopez LC, Harper ME, Saunders GF. Localization of the human glucagon gene (GCG) to chromosome segment 2q36-37. Cytogenet Cell Genet 1984;38:76-79. 30. Lebo RV, Lynch ED, Wiegant J, et al. Multicolor fluorence in situ hybridization cytogenetically orders gene loci. Genomics (submitted).
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DNA tumour virus-specific DNA sequences in abnormal human cervical biopsies by in situ hybridization. J Gen Vii01 1981;55:123-37. 43. Ambinder RF, Charach P, Staal S, et al. The Vector homology problem in diagnostic nucleic acid hybridization of clinical specimens. J Clin Microbial 1986;24:16-20. 44. Croen KD, Ostrove JM, Dragovic LJ, et al. Latent herpes simplex virus in human trigeminal ganglia. N Engl J Med 1987;317:23, 1427-32.
31. Lebo RV, Flandermeyer RR, Golbus M. Multicolor fluorescence in situ hybridization detects the most common aneuploidies. Am J Obstet and Gyn (submitted).
45. Shaw GM, Hahan BH, Arya SK, et al. Molecular characterization of human T-cell leukemia (lymphotrophic) virus type III in the acquired immune deficiency syndrome. Science 1984;226:1165-71.
32. Brigati DJ, Myerson D, Leary JJ, et al. Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes. Virology 1983;126:32-50.
46. Seiki M, Hattori S, Hirayama Y, Yoshida M. Human adult T-cell leukemia virus: Complete nucleotide sequence of the provirus genome integrated in leukemia cell DNA. Proc Nat1 Acad Sci USA 1983;80:3618-22.
33. Zachow KR, Ostrow RS, Bender M, et al. Detection of human papillomavirus DNA in anogenital neoplasia. Nature 1982;300:771-73.
47. Poeisz BJ, Morre JL, Mei S, et al. Biology and epidemiology of human T-cell leukemia lymphoma virus. In Gallo RC, Essex M, Gross L, eds. Human T-cell Leukemia-Lymphoma Virus: The Family of Human T-lymphotropic Retroviruses; Their Role in Malignancies and Association with AIDS. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 1984, pp 237-45.
34. Crum Cl’, Nagai N, Levin RLJ, Silverstein S. In situ hybridization analysis of HPV 16 DNA sequences in early cervical neoplasia. Am J Path01 1986;123:174-82. 35. Gupta J, Genelman HE, Naghashfar Z, et al. Specific identification of human papillomavirus types 11, 16 and 18 in lesions of the cervical smears and paraffin sections by in situ hybridization with radioactive probes; a preliminary communication. Int J Gynecol Path01 1985;4:211- 18. 36. Beckmann AM, Myerson D, Daling JR, et al. Detection and localization of human papillomavirus DNA in human genital condyloma by in situ hybridization with biotinylated probes. J Med Virol 1985;16:265-73. 37. Neumann R, Heiles B, Zippel C, et al. Use of biotinylated DNA probes in screening cells obtained from cervical swabs for human papillomavirus DNA sequences. Acta Cytologica 1986;30:6,603-07. 38. Adler-Storthz K, New1 and JR, Tessin BA, et al. Human papillomavirus DNA type 2 DNA in oral verrucous carcinoma. J Oral Pathol 1986;15:472-75. 39. Dehmezian RH, Batsakis JG, Goepfert H. In situ hybridization of papilloma virus DNA in head and neck squamous cell carcinomas. Arch Otolaryngol Head and Neck Surg 1987;113:819-21. 40. Magee KE, Rapini RF’, Duvic M, Storthz K. Human papilkeratoacanthoma. Arch Dermatol loma virus in 1989;125:1583-89. 41. Gendelman HE, Koenig S, Aksamit A, Venkatesan S. In situ hybridization for detection of viral nucleic acid in cell cultures and tissues. In Uhl GR, ed. Situ Hybridization in Brain. New York, Plenum Press, 1986, pp 203-23. 42. Maitland NJ, Kinross JH, Busuttil A, et al. The detection of
48. Bhagavati S, Ehrlich G, Kula RW, et al. Detection of human T-cell lymphoma/leukemia virus type I DNA and antigen in spinal fluid and blood of patients with chronic progressive. New Engl J Med 1988;318:1141-47. 49. Philips PE, Johnson SL, Runge LA, et al. High IgM antibody to human T-lymphotropic virus type I in systemic lupus erythematosus. J Clin Immuno 1986;6:234-41. 50. Zucker-Franklin D, Coutavas E, Zouzias D. Retrovirus infection of blood lymphocytes in patients with mycosis fungoides (abstr). Clin Res 1990;38:283A. 51. Ho D, Moudgil T, Masud A. Quantitation of human immunodeficiency virus type I in the blood of infected persons. N Engl J Med 1989;321:1621-25, 52. Richman DD, McCutchan JA, Spector SA. Detection of human immunodeficiency virus RNA in peripheral blood mononuclear cells by nucleic acid hybridization. J Infect Dis 1987;5:823-27. 53. Shaw GM, Harper ME, Hahan BH, et al. HTLV-III infection in brains of children and adults with AIDS encephalopathy. Science 1985;227:177-82. 54. Zucker-Franklin D, Yunzhen C. Megakaryocytes of human immunodeficiency virus-infected individuals express viral RNA. Proc Nat1 Acad Sci USA 1989;86:5595-99. 55. Ehrlich GA, Greenburg S, Abbott MA. Detection of human T-cell lymphoma/leukemia viruses. In Innis MA, Gelfand DH, Sninsky JJ, White TJ, ed. PCR Protocols. San Diego, Academic Press, Inc. 1990, pp 325-36
A
lthough several molecular biology techniques can be used to measure mRNA, only in situ hybridization (or in situ transcription) permit specific localization of DNA or an mRNA species within a tissue section or cell preparation. With appropriate fixation, mRNAs can be preserved and detected in tissue sections by using DNA or RNA probes labeled with radioactive or chemically modified nucleotides. In situ hybridization has been widely used in molecular and developmental biology to understand gene regulation in tissues and to localize the position of cloned genes on chromosomes, With the introduction of non-isotopic labeling techniques and commercial kits, in situ can be useful for detecting viral RNA or DNA or other genes of interest (like cancer-related oncogenes) in tissue specimens. The development of in situ hybridization is to molecular biology as immunofluorescence and immunoperoxidase staining have been to protein biochemistry, enabling localization and temporal relation of specific mRNAs .
Background In order to perform in situ hybridization, one must first isolate a probe for the gene of interest. Most commonly, this has been a cDNA complementary to the messenger RNA. cDNA isolation and cloning is a time-consuming and sometimes difficult project, which precedes one’s ability to do in situ hybridization, unless the cDNA has already been cloned. Hybridization of one strand of nucleic acid to another, whether RNA or DNA, depends on naturally occurring From the Department of Dermatology and internal Medicine, Dermatology Section, The University of Texas Medical School, M.D. Anderson Cancer Center, Houston, Texas. Address correspondence to: Madeleine Duvic, M.D., Department of Dermatology and Internal Medicine, M.D. Anderson Cancer Center, Houston, TX 77030.
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complementary base pairing between cytosine and guanine and between adenosine and either thymidine (DNA) or uracil (RNA). Heating cDNA melts the complementary strands, creating two single-stranded DNAs (called probes) that reanneal with DNA-complementary sequence to give a signal, Likewise, one can detect singlestranded RNA by the appropriate anti-sense complementary DNA single strand or by producing an anti-sense complementary RNA (riboprobe). In the standard Southern’ and Northern hybridizatier? techniques, DNA or RNA is first purified from tissue, separated by fragment size using electrophoresis, and transferred to a membrane for liquid hybridization with labeled probes. Both Southern and Northern hybridizations require the isolation of large (microgram) quantities of nucleic acids. In the isolation procedure, low copy number genes or RNAs are diluted, sometimes below the level of detection for the blotting techniques. By measuring nucleic acids in situ one may detect as little as one copy of a nucleic acid sequence. In situ hybridization techniques were first adapted to preparations of cells and chromosomes3T4 and used to detect amplified genes. 3,5,6Improvements in tissue fixation and labeling have made it possible to detect single copies of viral genomes in cells3 and single genes on chromosomes.‘,* Thus, many research applications of in situ hybridization include developmental and tumor biology, virology and microbiology, gene mapping and expression. Clinically, it has applications for improved cytogenetics and detection of genetic disease, cancer, and infectious organisms.9
Technical Aspects of In Situ Hybridization The basic method for in situ hybridization is shown in Figure 1. Included are a number of steps that differ slightly depending on the kind of probe used, how it will be labeled, and whether DNA or mRNA is to be detected.
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rrq 7. Wash/Remove
[r-G-T--] excess Probe
8
mpI”
Brn”lSlO”
(1 10 View and photograph
9 Autoradlogrwh
Figure
1.
preparations are dehydrated through alcohols and embedded in paraffin. Skin sections should be cut at 4 - 5 ,um thickness or less to avoid high background and trapping of probe. In some cases, to facilitate probe penetration, the tissue sections may be digested with a limited proteinase K digestion step or with HCl, heat, or detergent.3*10-14 Protease treatment may be more critical with the use of a glutaraldehyde-based fixative.3 The goal of tissue fixation is to optimally preserve the morphology of the tissues while stabilizing the mRNA and making it most accessible to the hybridization probe.
Outline of the in situ hybridization procedure.
C. Probe Preparation Excellent overviews of the procedure and its permutations include the following review articles.3*10-*2
A. Slide Preparation Tissue specimens are subjected to repeated washings and will often become detached unless special procedures are taken to make slides used more adherent. Soaking tissue sections in Elmer’s glue diluted in the waterbath prior to slide application works well for paraffin-embedded tissue used for DNA hybridization. However, for hybridization to mRNA, all contaminating RNase enzymes must be destroyed on the slides. We use the procedure adapted from Cox et a113-15 in which slides are washed in 10% Extran 300 overnight, washed exhaustively, and baked at 160°C. The slides are then coated with 2% 3-aminopropyltriethyloxysilane (Sigma) in acetone followed by RNase free water and dried at 42”C.15
B. Tissue Preparation
and Preservation
For detection of DNA (such as papilloma virus genome), routine fixation with buffered formalin followed by routine paraffin embedding and fixation is adequate.16*” As mRNAs are extremely labile and degraded by ubiquitous enzymes (RNases), preservation of RNA is critical. All equipment and glassware must be specially treated or sterile plastic used, and gloves must be used. Tissue can be snap frozen in liquid nitrogen and later cryosectioned, then fixed on the slide in paraformaldehyde according to the method of Harper.16 Alternatively, immediate fixation in 4% freshly made paraformaldehyde, which cross-links the mRNA just enough to allow excellent probe penetration with retention of the tissue morphology,14 can be used. Other fixatives that have been used for in situ hybridization include glutaraldehyde, formaldehyde, ethanol, or methanol.3J0-14 Following fixation, the tissue or cytospin
and Labeling
DNA Probes Single-stranded cDNA probes can be synthesized using phage Ml3 vectors or from viral RNA templates using reverse transcriptase. Cloned cDNA probes can be labeled by random primer method19 and melted to create single-stranded probes. In the latter case, there is competition between the annealing of the two complementary probe strands with the nucleic acid to be detected by in situ method.
RNA Probes A major improvement has been the development of plasmid vectors (pGEM, Promega, and others) containing Sp6 and T7 viral RNA polymerase promoters. These promoters flank a cassette of multiple restriction enzymes, which can be used to insert a cDNA of interest. Using the two promoters, single-stranded riboprobes are transcribed in opposite directions to generate sense and antisense strands that serve as negative and positive probes for mRNA molecules.15 These riboprobes are conveniently labeled during the transcription procedure with radioactive or biotinylated uridine triphosphate (UTP). Transcription and cloning kits are now available commercially. There are several major advantages to the riboprobes for in situ hybridization. Large microgram quantities of probe can be labeled to high specific activity. Following hybridization, unbound single-stranded probe can be removed by RNase treatment. RNase treatment plus the higher thermal stability of RNA-RNA duplexes compared to DNA-RNA hybrids allow higher washing temperatures, and reduce the non-specific background hybridization. For optimal penetration of riboprobes, reduction to 100 - 200 nucleotides is suggested in some protocols. This is achieved by limited alkaline lysis.13J4
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IN SITU HYBRIDIZATION
Control Probes Proper in situ hybridization technique demands the inclusion of control probes to demonstrate that binding is specific and not due to vector sequence or to non-specific hybridization. Riboprobes transcribedinoppositeorientations serve this purpose for detection of mRNA by the antisense strand. For detection of viral nucleic acids, it is important to include specimens known to be positive by other detection methods and to use heterologous probes from viruses not expected to be encountered. In some cases, it may be necessary to show that mRNA in the sample has not been degraded using a probe for a constituitively or abundantly expressed mRNA.
D. Hybridization
Conditions
During the hybridization step, the probe is allowed to anneal to the DNA or RNA within the specimen. The temperature, which is influenced by type, length, and sequence of probe used, is a critical parameter. Optimum temperature has been suggested as 25 “C below the melting temperature of the hybrids formed.14 The use of formamide in the reaction allows hybridization at lower temperatures but formamide is light sensitive. Hybridization is best performed under a coverslip in a humidified air chamber. Special hybridization “Probe-On” slides have been suggested by some to give superior results.*O To avoid high background, the probe concentration should be the lowest possible to still saturate the target mRNAs in a reasonable amount of time.3 Generally, a hybridization time of under 4 hours is recommended to keep background low. For a discussion of the reaction kinetics, the reader is referred to several reviews.3J0-14 The probe, if initially doublestranded, competes with its other strand for annealing to RNAs. Following the hybridization, excess unbound probe is removed by a series of stringency washes using excess volume of sodium citrate buffer and sodium dodecylsulfate. If riboprobes were used, unbound probes are digested with RNase during the wash procedure.i3,14
E. Detection Systems Radioactive Probes These have been most commonly used for in situ hybridization. Following washing to remove unbound probe, the slides are dipped in Kodak NTB2 emulsion, air dried, and autoradiographed at 4”C-70°C for a period of several days to weeks. t* The slides are then developed and fixed as by standard photography and analyzed by light and dark field microscopy. The number of grains per cell minus the background can be quantitated using a digitizer. Because of the problems interpreting a positive signal from background hybridization, we have used con-
Figure 2. In situ hybridization using %-UTP-labeled riboprobe to detect cells infected with the human immunodeficiency virus (HIV). (top): Bright field. (middle): Dark field. (bottom): Same field analyzed using confocal microscopy detectionz’Jz shows marked reduction in non-specific background.
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focal microscopy with digital image processing to determine specific hybridization signals.21*22 Figure 2 shows cells hybridizing with HIV riboprobe as detected by light microscopy (Figure 2A) and dark field (2B). The use of the confocal microscope results in elimination of the nonspecific background hybridization (Figure 2C). The choice of radioisotopes for in situ hybridization includes tritium 3H-, 32P-, and 35S-substituted nucleotide triphosphates. 3H is the most stable and gives the best localization with the lowest background signal but may take several months of autoradiography to give a detectable signal. 32P gives a strong sign al in the shortest period of time, but gives high background and poor localization. Most investigators use 35S as a compromise between the other two isotopes because it gives high specific activity and reasonable background.
Non-lsotopic Detection Alternative detection methods use cytochemical detection reactions, especially biotinylation with avidin-labeled detection using enzymatic or fluorescent molecules as reporters. Horseradish peroxidase and alkaline phosphatase are commonly used for enzymatic detection. Although these detection systems may be as sensitive as radionucleotides for detecting low-copy-number RNAs,‘O*” it is difficult, if not impossible, to quantitate amounts of mRNA detected at the present time. The advantage of cytochemical detection is the short period of time required for the analysis, compared to autoradiography, and the stability of probes, making them useful for clinically based diagnostics.
Applications of In Situ Hybridization Development
Biology
The advantage of in situ hybridization as a technique is its ability to localize low copy numbers of a specific mRNA or DNA molecule in a cell or tissue. In situ can be used to understand the timing and patterns of gene expression in embryogenesis, tissue differentiation, and disease. This is perhaps its most important research application at the current time. Cancer Biology In situ hybridization can be used to detect the breakpoint in tumor-related translocations, as well as to detect expression of cancer-related oncogenes. These can also be detected using Southern blotting and the more sensitive Polymerase Chain Reaction (PCR).
Gene Mapping The cloning of cDNAs for specific genes has been followed by an intense effort to map their location on chro-
mosomes. This can be best accomplished by a combination approach where the gene is localized to a specific chromosome, using somatic cell panels, and then to a specific chromosome band, using in situ hybridization.25,26 Somatic cell hybrid panels are generated by fusing animal (usually Chinese Hamster ovary cells) with human cells and selecting hybrid cells that contain one or more human chromosomes, or parts of chromosomes.25 DNA from each hybrid cell line is prepared and hybridized to labeled cDNA probes by standard Southern hybridization.’ By analyzing which lines hybridize to the cDNA probe, discordancy to the human chromosomes can be determined.25,26 When fractions of human chromosomes are found within hybrids, more specific localization to long or short arms can be made. Once the chromosome localization is determined, in situ hybridization can be a powerful tool to find the most probable gene location within one or more bands9r2’ For this procedure, white blood cells are stimulated to divide with phytohemaglutanin (PHA) and metaphase chromosomes are isolated and banded. In situ hybridization with 3H-labeled cDNA probes is then performed and analyzed by microscopy following autoradiography. The number of grains hybridizing to each chromosome are plotted from a number of cell preparations.27 Numerous genes have been mapped using this technique.27-29
Cytogenetics A technical advance in in situ hybridization has recently been developed that will enable researchers to determine the orientation of genes along one chromosome fragment.30 By using biotinylated cDNA probes detected by immunofluorescence dyes of different colors, it has been possible to orient several genetic markers along a stretch of a chromosome and therefore to determine probable gene order. 3oBecause in situ hybridization using immunofluorescent probes is so quick, and can be accomplished in 1 - 2 days, it may also become a very important cytogenetic tool with clinical application for determining chromosomal aneuploidy, such as Down’s Syndrome, in specimens collected by chorionic villus sampling or by amniocentesis.30,31 Detection of genetic disease may be accomplished in diseases in which large pieces of DNA are deleted, such as Duchenne’s muscular dystrophy and X-linked ichthyosis. In Situ Hybridization
Can Detect Viral Nucleic Acids
One of the earliest clinical applications of in situ hybridization was to detect human viruses in cells and tissue specimens using biotinylated32 or radioactive DNA probes.3 Human papilloma viruses (HPV) are DNA viruses that have been implicated in the etiology of squa-
Clinics in Dermatology 1992;9:229-235 mous cell carcinomas, especially of the anogenital tract. Earliest studies for detection of viral DNA were dot-blot hybridizations using extracted DNA. However, dot-blots only tell whether a nucleic acid is present or absent and do not give information regarding which cells are infected.33 As HPV types 16 and 18 have been implicated as more carcinogenic, determination of the specific HPV type has been of clinical interest in studying specimens from cervical and anogenital carcinomas, and from condylomata by In addition to tissue speciin situ hybridization. 14~15,34-36 mens, cervical cells from Pap smears have also been studied for HPV DNA sequences and have shown a high prevalence of HPV 16 and 18 sequences in severe dysplasia and carcinoma.35,37 A viral detection kit for Pap smear in situ hybridization is commercially available. In situ hybridization has also been used to demonstrate the presence of HPV in oral carcinomas3* and recently in other squamous cell carcinomas of the head and neck39 and in keratoacanthomas.40 Typing of HPV by in situ hybridization, at least for the handful of more virulent strains, is a feasible, clinical application of the technique using biotinylated DNA probes in paraffin-embedded specimens or cytology preparations, and is more sensitive than routine Pap smears in detecting HPV. Other DNA viruses have also been detected using in situ hybridization. 41 Abnormal human cervical tumor biopsies were found to contain Herpes simplex type 2 (67%), and adenovirus type 2 (39%) using 3H-labeled DNA probes versus 23% and 17% of normal biopsies.42 However, because tissue specimens may contain bacterial contamination including plasmids, which cross hybridize with the vector used to make probes,43 the appropriate vector control probe should be used and give a low percentage of positive signals.42 The mechanism of how herpes simplex virus remains latent in the ganglia was studied in trigeminal ganglion from autopsies using in situ hybridization.M Positive signals were detected in 67% of subjects, similar to the proportion of seropositive individuals. Interestingly, an immediate early gene (ICPO), which is actively expressed during acute infection, was expressed as an antisense transcript, suggesting that antisense RNA may maintain latency of the virus. 44 The power of in situ hybridization for virology is that it will detect provirus incorporated into the DNA or in the cytoplasm in the absence of viral protein or particle expression. This ability to detect latent virus is already changing our concept of the role of viruses in human diseases. Retroviruses are RNA viruses with the capability of incorporating themselves into host DNA using the enzyme reverse transcriptase. They can remain latent in cells without expressing virus or viral particles for long periods of time, and can be inherited through the germ-
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line-like genes. In situ hybridization as well as PCR can be used to detect latent or low copy-number retrovirus infections.3 Standard Southern hybridization techniques, on the other hand, cannot identify nucleic acid sequences present in amounts less than one copy for every 10 - 100 cells.45 The role of retroviruses in causing human diseases has only recently been appreciated with the discovery of HTLV-I in T-cell lymphoma cells.46,47 HTLV-I has also been associated with tropical spastic paraparesis,‘* and is less well documented in association with systemic lupus erythematosus,49 and mycosis fungoides (cutaneous T cell lymphoma).50 The human immunodeficiency virus HIV-I (formerly HTLV-III), is another recently discovered retrovirus of the lentivirus family that infects and destroys the human immune system, causing AIDS and/or severe neurologic dysfunction. 45 In situ hybridization has been used to study the cell populations infected by HIV, including CD4+ T cells,18,51 monocytes,52 microglial cells,53 megakaryocytes,54 and dermal dendritic cells.*l The pattern of infection of cells with HIV may well explain the manifold clinical manifestations associated with this virus,
Comparison of In Situ Hybridization with the Polymerase Chain Reaction The polymerase chain reaction (PCR) is also being widely used to detect viral nucleic acids in cells and tissue.55 As it is many times more sensitive than in situ hybridization and more rapid to perform, it may turn out to be the more clinically useful detection method. However, the sensitivity of PCR may lead to a high incidence of false positives. Furthermore, PCR does not give information regarding the localization of nucleic acids unless performed on single cells or sperm. PCR is the most sensitive detection method possible because of its capability to amplify one copy of DNA many millions of times. Although PCR can also be used to detect mRNA following production of a first-strand cDNA species, and may be made quantitative with the use of an internal standard, this latter procedure is a multistep process that may not lend itself to a routine laboratory test. Thus, in situ hybridization may continue to have clinical applications where cellular or temporal localization of one or more nucleic acids are desired, or where simultaneous detection of protein and mRNAs are required to be visualized. However, in situ hybridization does not easily lend itself to quantitation, especially using enzymatic or fluorescent detection methods. Because in situ hybridization using conventional radioactive probes is a technique requiring fastidious conditions and weeks to perform, it is unlikely to be widely adapted for clinical use. However,
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with the development of commercially available probes labeled non-isotopically for fluorescent or enzymatic reporter detection, clinical applications of the technique may become much more widely used for screening or detection of medically important mRNAs or cytogenetic abnormalities, and of nucleic acids from infectious agents that would be impossible to detect by other means. Acknowledgments This work was supporfed in part by NIH grants R29AR36546 and ROl-AR39975. The author wishes to thank Elaine Fuchs, Ph.D. and Andrea Sfoler, Ph.D. for their help, and acknowledges the research collaboration of Wanda T. Schroeder, Ph.D., Sfeven Mahoney, M.D., and Wanda O’Brien, B.A. Dr. Schroederwas the recipient ofDermatology Foundation Fellowships from Roche Laboratories and Squibb Pharmaceuticals. Dr. Mahoney was the recipient ofa Dermatology Fellowship from Beecham Laboratories.
References 1. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975;89:50317. 2. Thomas PS. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Nat1 Acad Sci USA 1974;71:5201-05. 3. Haase A, Brahic M, Stowring L, Blum H. Detection of viral nucleic acids by In Situ hybridization. In Methods in Virology, New York Academic Press, Inc., 1984, pp 212- 14. 4. John HA, Birnstiel ML, Jones KW. RNA-DNA hybrids at the cytological level. Nature 1969;223:582-87.
12. Gee CE, Roberts JL. In situ hybridization histochemistry: A technique for the study of gene expression in single cells. DNA 1983;2:157-63. 13. Cox KH, DeLeon DV, Angerer LM, Angerer RC. Detection of mRNAs in sea urchin embryos by in situ hybridization suing asymmetric RNA probes. Dev Bioll984,485-502. 14. Angerer RC, Cox KH, Angerer LM. In situ hybridization to cellular RNAs. In Setlow JK, Holleander A, eds. Genetic Engineering, Vol. 7. New York and London, Plenum Press, 1985, pp 43-65. 15. Stoler A, Kopan R, Duvic M, Fuchs E. Use of monospecific antisera and cRNA probes to localize the major changes in keratin expression during normal and abnormal epidermal differentiation. J Cell Biol 1988;107:427-46. 16. Wells M, Griffiths S, Lewis F, Bird C. Demonstration of human papillomavirus types in paraffin processed tissue from human anogenital lesions by in situ DNA hybridization. J Path01 1987;152:77-82. 17. von Krogh G. Advantage of human papilloma virus typing in the clinical evaluation of genitoanal warts. Experience with the in situ deoxyribonucleic acid hybridization technique applied on paraffin sections. J Am Acad Dermatol 1988;18:495-503. 18. Harper ME, Marselle LM, Gallo RC, Wong-Staal F. Detection of lymphocytes expression human T-lymphotrophic virus type III in lymph nodes and peripheral blood from infected individuals by in situ hybridization. Proc Nat1 Acad Sci USA 1986;83:772-76. 19. Feinberg AI’, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1984;137:266-67. 20. Munjaal RF’, Conneely OM, O’Malley BW. In situ detection of progesterone receptor mRNA in the chicken oviduct using probe-on slides. BioTechniques 1989;7:1104-07.
5. Jones KW. Chromosomal and nuclear location of mouse satellite DNA in individual cells. Nature 1970;225:91215.
21. Mahoney SE, Duvic M, Nickoloff BJ, et al. HIV transcripts identified in HIV-related psoriasis and Kaposi’s sarcoma lesions. J Clin Invest (submitted).
6. Henderson AS, Warburton D, Atwood KC. Location of ribosomal DNA in the human chromosome complement. Proc Nat1 Acad Sci USA 1972;69:3394-98.
22. Shotten DM. Confocal scanning optical microscopy and its applications for biological specimens. J Cell Sci 1989;94:175-206.
7. Tereba A, Lai MMC, Murti KG. Chromosome 1 contains the endogeneous RAV-0 retrovirus sequences in chicken cells. Proc Nat1 Acad Sci USA 1979;76:6486-90.
23. Venezky DL, Angerer IM, Angerer RC. Accumulation of histone repeat transcripts in the urchin egg pronuclease. Cell 1981;24:385-91.
8. Harper ME, Ulrich A, Saunders GF. Localization of the human insulin gene to the distal end of the short arm of chromosome 11. Proc Nat1 Acad Sci USA 1981;78: 4458-60.
24. Berman SA, Bursztajn S, Bowen B, Gilbert W. Localization of an acetylcholine receptor intron to the nuclear membrane. Science 1990;247:212-14.
9. Lichter I’, Ward DC. Is non-isotopic in situ hybridization finally coming of age? Nature 1990;345:93-94. 10. Singer RH, Lawrence JB, Villnave C. Optimization of in situ hybridization using isotopic and non-isotopic detection methods. BioTechniques 1986;4:230-45. 11. Bresser J, Evinger-Hodges MJ. Comparison and optimatization of in situ hybridization procedures yielding rapid, sensitive mRNA detections. Gene Anal Techn 1987;4:89- 104.
25. Thompson LH, Carrano AV, Sato K, et al. Identification of nucleotide-excision repair genes on human chromosomes 2 and 13 by functional complementation in hamster-human hybrids. Somat Cell Molec Genet 1987;13:539-51. 26. Sicilian0 MJ, Carrano AV, Thompson LH. Assignment of a human DNA repair gene associated with sister chromatid exchange to chromosome 19. Mutation Res 1986;174: 303-08. 27. Harper ME, Saunders GF. Localization of single copy DNA
Clinics in Dermatology 7991;9:129-135
sequences on G banded chromosomes by in situ hybridization. Chromosoma 1981;83:431-39. 28. Human Gene Mapping 9. In Klinger HP, ed. Cytogenetics and Cell Genetics. Basel, S. Karger, 1987, p 46. 29. Schroeder WT, Lopez LC, Harper ME, Saunders GF. Localization of the human glucagon gene (GCG) to chromosome segment 2q36-37. Cytogenet Cell Genet 1984;38:76-79. 30. Lebo RV, Lynch ED, Wiegant J, et al. Multicolor fluorence in situ hybridization cytogenetically orders gene loci. Genomics (submitted).
DUVIC IN SITU HYBRIDIZATION
135
DNA tumour virus-specific DNA sequences in abnormal human cervical biopsies by in situ hybridization. J Gen Vii01 1981;55:123-37. 43. Ambinder RF, Charach P, Staal S, et al. The Vector homology problem in diagnostic nucleic acid hybridization of clinical specimens. J Clin Microbial 1986;24:16-20. 44. Croen KD, Ostrove JM, Dragovic LJ, et al. Latent herpes simplex virus in human trigeminal ganglia. N Engl J Med 1987;317:23, 1427-32.
31. Lebo RV, Flandermeyer RR, Golbus M. Multicolor fluorescence in situ hybridization detects the most common aneuploidies. Am J Obstet and Gyn (submitted).
45. Shaw GM, Hahan BH, Arya SK, et al. Molecular characterization of human T-cell leukemia (lymphotrophic) virus type III in the acquired immune deficiency syndrome. Science 1984;226:1165-71.
32. Brigati DJ, Myerson D, Leary JJ, et al. Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes. Virology 1983;126:32-50.
46. Seiki M, Hattori S, Hirayama Y, Yoshida M. Human adult T-cell leukemia virus: Complete nucleotide sequence of the provirus genome integrated in leukemia cell DNA. Proc Nat1 Acad Sci USA 1983;80:3618-22.
33. Zachow KR, Ostrow RS, Bender M, et al. Detection of human papillomavirus DNA in anogenital neoplasia. Nature 1982;300:771-73.
47. Poeisz BJ, Morre JL, Mei S, et al. Biology and epidemiology of human T-cell leukemia lymphoma virus. In Gallo RC, Essex M, Gross L, eds. Human T-cell Leukemia-Lymphoma Virus: The Family of Human T-lymphotropic Retroviruses; Their Role in Malignancies and Association with AIDS. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 1984, pp 237-45.
34. Crum Cl’, Nagai N, Levin RLJ, Silverstein S. In situ hybridization analysis of HPV 16 DNA sequences in early cervical neoplasia. Am J Path01 1986;123:174-82. 35. Gupta J, Genelman HE, Naghashfar Z, et al. Specific identification of human papillomavirus types 11, 16 and 18 in lesions of the cervical smears and paraffin sections by in situ hybridization with radioactive probes; a preliminary communication. Int J Gynecol Path01 1985;4:211- 18. 36. Beckmann AM, Myerson D, Daling JR, et al. Detection and localization of human papillomavirus DNA in human genital condyloma by in situ hybridization with biotinylated probes. J Med Virol 1985;16:265-73. 37. Neumann R, Heiles B, Zippel C, et al. Use of biotinylated DNA probes in screening cells obtained from cervical swabs for human papillomavirus DNA sequences. Acta Cytologica 1986;30:6,603-07. 38. Adler-Storthz K, New1 and JR, Tessin BA, et al. Human papillomavirus DNA type 2 DNA in oral verrucous carcinoma. J Oral Pathol 1986;15:472-75. 39. Dehmezian RH, Batsakis JG, Goepfert H. In situ hybridization of papilloma virus DNA in head and neck squamous cell carcinomas. Arch Otolaryngol Head and Neck Surg 1987;113:819-21. 40. Magee KE, Rapini RF’, Duvic M, Storthz K. Human papilkeratoacanthoma. Arch Dermatol loma virus in 1989;125:1583-89. 41. Gendelman HE, Koenig S, Aksamit A, Venkatesan S. In situ hybridization for detection of viral nucleic acid in cell cultures and tissues. In Uhl GR, ed. Situ Hybridization in Brain. New York, Plenum Press, 1986, pp 203-23. 42. Maitland NJ, Kinross JH, Busuttil A, et al. The detection of
48. Bhagavati S, Ehrlich G, Kula RW, et al. Detection of human T-cell lymphoma/leukemia virus type I DNA and antigen in spinal fluid and blood of patients with chronic progressive. New Engl J Med 1988;318:1141-47. 49. Philips PE, Johnson SL, Runge LA, et al. High IgM antibody to human T-lymphotropic virus type I in systemic lupus erythematosus. J Clin Immuno 1986;6:234-41. 50. Zucker-Franklin D, Coutavas E, Zouzias D. Retrovirus infection of blood lymphocytes in patients with mycosis fungoides (abstr). Clin Res 1990;38:283A. 51. Ho D, Moudgil T, Masud A. Quantitation of human immunodeficiency virus type I in the blood of infected persons. N Engl J Med 1989;321:1621-25, 52. Richman DD, McCutchan JA, Spector SA. Detection of human immunodeficiency virus RNA in peripheral blood mononuclear cells by nucleic acid hybridization. J Infect Dis 1987;5:823-27. 53. Shaw GM, Harper ME, Hahan BH, et al. HTLV-III infection in brains of children and adults with AIDS encephalopathy. Science 1985;227:177-82. 54. Zucker-Franklin D, Yunzhen C. Megakaryocytes of human immunodeficiency virus-infected individuals express viral RNA. Proc Nat1 Acad Sci USA 1989;86:5595-99. 55. Ehrlich GA, Greenburg S, Abbott MA. Detection of human T-cell lymphoma/leukemia viruses. In Innis MA, Gelfand DH, Sninsky JJ, White TJ, ed. PCR Protocols. San Diego, Academic Press, Inc. 1990, pp 325-36
