Skip to main content
PMC home page
Journal of Clinical Laboratory Analysis logoLink to Journal of Clinical Laboratory Analysis

In situ hybridization: Methods and applications

Long Jin 1, Ricardo V Lloyd 1,✉
PMCID: PMC6760707  PMID: 9021518

Abstract

In situ hybridization (ISH) combines molecular biological techniques with histological and cytological analysis of gene expression. RNA and DNA can be readily localized in specific cells with this method. ISH has been useful as a research tool, and recent studies have used this technique in the diagnostic pathology laboratory and in microbiology for the tissue localization in infectious agents.

Other recent developments in the applications of ISH involve in situ polymerase chain reaction (PCR) and in situ reverse transcription (RT)‐PCR, which can be used to detect very low levels of nucleic acids in tissues by taking advantage of the powerful amplification capacity of PCR. In situ PCR will contribute significantly to progress in this field because of the marked increase in the sensitivity of this method. J. Clin. Lab. Anal. 11:2–9. © 1997 Wiley‐Liss, Inc.

Keywords: in situ hybridization, polymerase chain reaction, in situ polymerase chain reaction

References

  • 1. Gall JG, Pardue ML: Formation and detection of RNA‐DNA hybrid molecules in cytological preparation. Proc Natl Acad Sci USA 63: 378–383, 1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Egger D, Troxler M, Bienz K: Light and electron microscopic in situ hybridization: Non‐radioactive labeling and detection, double hybridization, and combined hybridization‐immunocytochemistry. J Histochem Cytochem 42: 815–822, 1994. [DOI] [PubMed] [Google Scholar]
  • 3. Wilcox JN: Fundamental principles of in situ hybridization. J Histochem Cytochem 41: 1725–1733, 1993. [DOI] [PubMed] [Google Scholar]
  • 4. Wilkinson DG, ed: In Situ Hybridization: A Practical Approach. IRL Press—Oxford University Press, New York, 1993. [Google Scholar]
  • 5. Panoskaltsis‐Mortari A, Bucy RP: In situ hybridization with digoxigenin‐labeled RNA probes. Facts and artifacts. Biotechniques 18: 300–307, 1995. [PubMed] [Google Scholar]
  • 6. DeLellis RA: In situ hybridization techniques for the analysis of gene expression: Applications in tumor pathology. Hum Pathol 25: 580–585, 1994. [DOI] [PubMed] [Google Scholar]
  • 7. Lloyd RV, Jin L: In situ hybridization analysis of chromogranin A and B mRNAs in neuroendocrine tumors with digoxigenin‐labeled oligonucleotide probe cocktails. Diagn Mol Pathol 4: 143–151, 1995. [DOI] [PubMed] [Google Scholar]
  • 8. Szakacs JG, Livingston SK: MRNA in‐situ hybridization using biotinylated oligonucleotide probes: Implications for the diagnostic laboratory. Ann Clin Lab Sci 24: 324–338, 1994. [PubMed] [Google Scholar]
  • 9. Brooks PJ, Kaplitt MG, Kleopoulos SP, Funabashi T, Mobbs CV, Peaff DW: Detection of messenger RNA and low‐abundance heteronuclear RNA with single‐stranded DNA probes produced by amplified primer extension labeling. J Histochem Cytochem 41: 1761–1766, 1993. [DOI] [PubMed] [Google Scholar]
  • 10. Komminoth P, Merk FB, Leav I, Wolfe HJ, Roth J: Comparison of 35Sand digoxigenin‐labeled RNA and oligonucleotide probes for in situ hybridization. Expression of mRNA of the seminal vesicle secretion protein II and androgen receptor genes in the rat prostate. Histochemistry 98: 217–228, 1992. [DOI] [PubMed] [Google Scholar]
  • 11. Lloyd RV, Cano M, Chandler WF, Barkan AL, Horvath E, Kovacs K: Human growth hormone and prolactin secreting pituitary adenomas analyzed by in situ hybridization. Am J Pathol 134: 605–613, 1989. [PMC free article] [PubMed] [Google Scholar]
  • 12. Lloyd RV, Jin L, Kulig E, Fields K: Molecular approaches for the analysis of chromogranins and secretogranins. Diagn Mol Pathol 1: 2–15, 1992. [DOI] [PubMed] [Google Scholar]
  • 13. Pagani A, Cerrato M, Bussolati G: Nonspecific in situ hybridization reaction in neuroendocrine cells and tumors of the gastrointestinal tract using oligonucleotide probes. Diagn Mol Pathol 2: 125–130, 1993. [PubMed] [Google Scholar]
  • 14. Weiss LM, Movahed LA, Chen YY, Shin SS, Stroup RM, Bui N, Estess P, Bindl JM: Detection of immunoglobulin light‐chain mRNA in lymphoid tissues using a practical in situ hybridization method. Am J Pathol 137: 979–988, 1990. [PMC free article] [PubMed] [Google Scholar]
  • 15. Stahl WL, Eakin TJ, Baskin DG: Selection of oligonucleotide probes for detection of mRNA isoforms. J Histochem Cytochem 41: 1735–1740, 1993. [DOI] [PubMed] [Google Scholar]
  • 16. Miller MA, Kolb PE, Raskind MA: A method for simultaneous detection of multiple mRNAs using digoxigenin and radioisotopic cRNA probes. J Histochem Cytochem 41: 1741–1750, 1993. [DOI] [PubMed] [Google Scholar]
  • 17. Farquharson MA, Harvie R, Kennedy A, McNicol AM: Detection of mRNA by in situ hybridization and in northern blot analysis using oligodeoxynucleotide probes labeled with alkaline phosphatase. J Clin Pathol 45: 999–1002, 1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Thomas GA, Davies HG, Williams ED: Demonstration of mRNA using digoxigenin labeled oligonucleotide probes for in situ hybridization in formamide free conditions. J Clin Pathol 46: 171–174, 1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Singer RH, Ward DC: Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with biotinated nucleotide analogue. Proc Natl Acad Sci USA 79: 7331–7335, 1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Yoshii A, Koji T, Ohsawa N, Nakome P: In situ hybridization of ribosomal RNAs is a reliable reference for hybridizable RNA in tissue sections. J Histochem Cytochem 43: 321–327, 1995. [DOI] [PubMed] [Google Scholar]
  • 21. Stoler MH, Rhodes CR, Whitbeck A, Wolinsky SM, Chow LT, Broker TR: Human papillomavirus type 16 and 18 gene expression in cervical neoplasia. Hum Pathol 23: 117–128, 1992. [DOI] [PubMed] [Google Scholar]
  • 22. Choi YJ: In situ hybridization using a biotinylated DNA probe on formalin‐fixed liver biopsies with hepatitis B‐virus infections: In situ hybridization superior to immunohistochemistry. Mod Pathol 3: 343–347, 1990. [PubMed] [Google Scholar]
  • 23. Ambinder RF, Mann RB: Epstein‐Barr‐encoded RNA in situ hybridization: Diagnostic applications. Hum Pathol 25: 602–605, 1994. [DOI] [PubMed] [Google Scholar]
  • 24. Chang KL, Chen YY, Shibata DS, Weiss LM: Description of an in situ hybridization methodology for detection of Epstein‐Barr virus RNA in paraffin‐embedded tissues with a survey of normal and neoplastic tissues. Diagn Mol Pathol 1: 246–255, 1992. [PubMed] [Google Scholar]
  • 25. Hamilton‐Dutoit SJ, Pallesen G: Detection of Epstein‐Barr virus small RNAs in routin paraffin sections using non‐isotopic RNA/RNA in situ hybridization. Histopathology 25: 101–111, 1994. [DOI] [PubMed] [Google Scholar]
  • 26. Montone KT, Litzky LA: Rapid method for detection of Aspergillus 5 g ribosomal RNA using a genus‐specific oligonucleotide probe. Am J Clin Pathol 103: 48–51, 1995. [DOI] [PubMed] [Google Scholar]
  • 27. Gray JW, Pinkel D, Brown JM: Fluorescence in situ hybridization in cancer and radiation biology (review). Radial Res 137: 275–289, 1994. [PubMed] [Google Scholar]
  • 28. Lloyd RV: Molecular probes and endocrine disease. Am J Surg Pathol 14(suppl): 34–14, 1990. [PubMed] [Google Scholar]
  • 29. Jin L, Chandles WF, Smart JB, England BG, Lloyd RV: Differentiation of human pituitary adenomas determines the pattern of chromogranin/secretogranin messenger ribonucleic acid expression. J Clin Endocrinol Metab 76: 728–735, 1993. [DOI] [PubMed] [Google Scholar]
  • 30. Varma VA, Cerjan CM, Abbott KL, Hunter SB: Non‐isotopic in situ hybridization method for mitochondria in oncocytes. J Histochem Cytochem 42: 273–276, 1994. [DOI] [PubMed] [Google Scholar]
  • 31. Kerstens HMJ, Poddighe PJ, Hanselaar AGJM: A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine. J Histochem Cytochem 43: 347–352, 1995. [DOI] [PubMed] [Google Scholar]
  • 32. Kriegsmann J, Keyszer G, Geiler T, Gay RE, Gay S: A new double labeling technique for combined in situ hybridization and immunohistochemical analysis. Lab Invest 71: 911–917, 1994. [PubMed] [Google Scholar]
  • 33. Trembleau A, Roche D, Galas A: Combination of non‐radioactive and radioactive in situ hybridization with immunohistochemistry: A new method allowing the simultaneous detection of two mRNAs and one antigen in the same brain tissue section. J Histochem Cytochem 41: 489–98, 1993. [DOI] [PubMed] [Google Scholar]
  • 34. Dagerlind A, Friberg K, Bean AJ, Hökfelt T: Sensitive detection using unfixed tissues: Combined radioactive and nonradioactive in situ hybridization histochemistry. Histochemistry 98: 39–43, 1992. [DOI] [PubMed] [Google Scholar]
  • 35. Young WS III, Hsu AC: Observations on the simultaneous use of digoxigenin and radiolabeled oligodeoxyribonucleotide probes for hybridization histochemistry. Neuropeptides 18: 75, 1991. [DOI] [PubMed] [Google Scholar]
  • 36. Van Wijk U, Van Vugt JMG, Könst AAM, Mulders MAM, Nieuwint AWM, Oudejans CBM: Multiparameter in situ hybridization of trophoblast cells in mixed cell populations by combined DNA and RNA in situ hybridization. J Histochem Cytochem 43: 709–714, 1995. [DOI] [PubMed] [Google Scholar]
  • 37. Bienz K, Egger D: Immunocytochemistry and in situ hybridization in the electron microscope: Combined application in the study of virus‐infected cells. Histochemistry 103: 328–338, 1995. [DOI] [PubMed] [Google Scholar]
  • 38. Lloyd RV, Jin L, Song J: Ultrastructural localization of prolactin and chromogranin B messenger ribonucleic acids with biotinylated oligonucleotide probes in cultured pituitary cells. Lab Invest 63: 413–19, 1990. [PubMed] [Google Scholar]
  • 39. Sibon OCM, Cremers FFM, Boonstra HJ, Verkleij: Localization of nuclear RNA by pre‐ and post‐embedding in situ hybridization using different gold probes. Histochem J 27: 35–5, 1995. [DOI] [PubMed] [Google Scholar]
  • 40. Bagasra O, Seshamma T, Hansen J, Bobroski L, Saikumari P, Pestaner JP, Pomerantz RJ: Application of in situ PCR methods in molecular biology. I. Details of methodology for general use. Cell Vision 1: 324–335, 1994. [Google Scholar]
  • 41. Long AA, Komminoth P: Study of viral DNA using in situ PCR. Cell Vision 1: 56–57, 1994. [Google Scholar]
  • 42. Komminoth P, Long AA: In situ polymerase chain reaction and its applications to the study of endocrine diseases. Endocr Pathol 6: 167–171, 1995. [Google Scholar]
  • 43. Embleton MJ, Gorochov G, Jones PT, Winter G: In‐cell PCR from mRNA: Amplifying and linking the rearranged immunoglobulin heavy and light chain V‐genes within single cells. Nucleic Acids Res 20: 3831–3837, 1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Haase AT, Retzel EF, Staskus KA: Amplification and detection of lentiviral DNAinside cells. Pro Natl Acad Sci USA 87: 4971–4975, 1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45. Nuovo GJ, Gallery F, MaConnell P, Becker J, Bloch W: An improved technique for the in situ detection of DNA after polymerase chain reaction amplification. Am J Pathol 139: 1239–1244, 1991. [PMC free article] [PubMed] [Google Scholar]
  • 46. Martinez A, Miller MJ, Quinn K, Unsworth EJ, Ebina M, Cuttitta F: Non‐radioactive localization of nucleic acids by direct in situ PCR and in situ RT‐PCR in paraffin‐embedded sections. J Histochem Cytochem 43: 739–747, 1995. [DOI] [PubMed] [Google Scholar]
  • 47. Long AA, Komminoth P, Lee E, Wolfe HJ: Comparison of indirect and direct in‐situ polymerase chain reaction in cell preparations and tissue sections. Detection of viral DNA, gene rearrangements, and chromosomal translocations. Histochemistry 99: 151–162, 1993. [DOI] [PubMed] [Google Scholar]
  • 48. Nuovo GJ, Lidonnici K, MacConnell P, Lane B: Intracellular localization of polymerase chain reaction (PCR)‐amplified hepatitis C cDNA. Am J Surg Pathol 17: 683–690, 1993. [DOI] [PubMed] [Google Scholar]
  • 49. Bibbo M, Pestanet JP, Scavo LM, Bobroski L, Seshamma T, Bagasra O: Surfactant protein A mRNA expression utilizing the reverse transcription in situ PCR for metastatic adenocarcinoma (case report). Cell Vision 1: 290–293, 1994. [Google Scholar]
  • 50. Jin L, Qian X, Lloyd RV: Comparison of mRNA expression detected by in situ polymerase chain reaction and in situ hybridization in endocrine cells. Cell Vision 2: 314–321, 1995. [Google Scholar]
  • 51. Chen RH, Fuggle SV: In situ cDNA polymerase chain reaction. A novel technique for detecting mRNA expression. Am J Pathol 143: 1527–1534, 1993. [PMC free article] [PubMed] [Google Scholar]
  • 52. Patel VG, Shum‐Siu A, Heniford BW, Wieman TJ, Hendler FJ: 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, 1994. [PMC free article] [PubMed] [Google Scholar]
  • 53. Bagasra O, Seshamma T, Hansen J, Bobroski L, Saikumari P, Pomerantz RJ: Application of in situ PCR methods in molecular biology. II. Special applications in electron microscopy. Cytogenetics and immunohistochemistry. Cell Vision 2: 61–70, 1995. [Google Scholar]

Articles from Journal of Clinical Laboratory Analysis are provided here courtesy of Wiley

RESOURCES