Summary
The sensitivity of radiolabeled and digoxigenin-labeled RNA probes and synthetic oligonucleotide probes for the detection of seminal vesicle secretion protein II (SVS II) and androgen receptor (AR) mRNA was compared by in situ hybridization in paraformaldehyde-fixed cryostat sections of the rat prostate. Both genes are expressed in different amounts in the various prostatic lobes and contiguous glands. SVS II or AR RNA probes were either labeled with digoxigenin-11-UTP or [35S]UTP by in vitro transcription. A synthetic SVS II oligonucleotide probe was 3′ end-labeled (tailed) with either digoxigenin-11-dUTP or [35S]dATP. Hybridized 35S-labeled probes were detected by autoradiography and digoxigenin-labeled probes by immunohistochemistry using alkaline phosphatase conjugated anti-digoxigenin antibody or gold-labeled antibody followed by protein A-gold and silver enhancement. Digoxigenin-labeled probes provided the same degree of sensitivity as their 35S-labeled counterparts for the detection by in situ hybridization of weakly and strongly expressed mRNA. Using both labeling methods, the SVS II RNA probes were more sensitive than the oligonucleotide probes and background labelling of the 35S-labeled oligonucleotide probe was high. The digoxigenin method produced less background with all probe types, hybridization signals showed higher resolution and results were obtained faster than with radiolabeled probes. The immunogold silver enhancement system provided the fastest detection of digoxigenin-labeled probes with a sensitivity and resolution similar to that provided by alkaline phosphatase anti-digoxigenin immunohistochemistry. It is concluded that digoxigenin probe labeling and detection provides a sensitive, reliable, and efficient alternative to radiolabeled probes for in situ hybridization of mRNA.
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References
Baldino F Jr, Lewis ME (1989) Nonradioactive in situ hybridization histochemistry with digoxigenin-deoxyuridine 5′-triphosphate-labeled oligonucleotides. In: Conn PM (ed) Gene probes. Methods in neuroscience. Academic Press, San Diego, pp 282–292
Chang C, Kokontis J, Liao S (1988) Structural analysis of complementary DNA and amino acid sequences of human and rat androgen receptors. Proc Natl Acad Sci USA 85:7211–7215
Cook AF, Voucolo E, Brakel CL (1988) Synthesis and hybridization of biotinylated oligonucleotides. Nucleic Acids Res 16:4077–4095
Dodd JG, Kreis Ch, Sheppard PC, Hamel A, Matusik RJ (1986) Effect of androgens on mRNA for a secretory protein of dorsolateral prostate and seminal vesicles. Mol Cell Endocrinol 47:191–200
Farquharson M, Harvie R, McNicol AM (1990) Detection of messenger RNA using a digoxigenin end labelled oligodeoxynucleotide probe. J Clin Pathol 43:424–428
Forster AC, McInnes JL, Skingle JC, Symons RH (1985) Nonradioactive hybridization probes prepared by chemical labeling of DNA and RNA with a novel reagent, photobiotin. Nucleic Acids Res 13:745–761
Furuta Y, Shinohara T, Sano K, Meguro M, Nagashima K (1990) In situ hybridization with digoxigenin-labelled DNA probes for detection of viral genomes. J Clin Pathol 43:806–809
Gall JG, Pardue ML (1971) Nucleic acid hybridization in cytological preparations. Methods Enzymol 38:470–480
Gerfen CR (1989) Quantification of in situ hybridization histochemistry for analysis of brain function. In: Conn PM (ed) Gene probes. Methods in neuroscience. Academic Press, San Diego, pp 79–97
Harris SE, Harris MA, Johnson CM, Bean MF, Dodd JG, Matusik RJ, Carr SA, Crabb JW (1990) Structural characterization of the rat seminal vesicle secretion II protein and gene. J Biol Chem 265:9896–9903
Heiles HBJ, Genersch E, Kessler C, Neumann R, Eggers HJ (1988) In situ hybridization with digoxigenin-labeled DNA of human papillomaviruses (HPV16/18) in HeLa and SiHa cells. BioTechniques 6:978–981
Heino P, Hukkanen V, Arstila P (1989) Detection of human papilloma viruses (HPV) DNA in genital biopsy specimens by in situ hybridization with digoxigenin-labeled probes. J Virol Methods 26:331–337
Höfler H, Childers H, Montminy MR, Lechan RM, Goodmann RH, Wolfe HJ (1986) In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using anti-sense RNA probes. Histochem J 18:597–604
Hopman AHN, Wiegant J, Van Duijn P (1986) A new hybridocyto-chemical method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands. I. Stability of mercury-sulfhydryl bond and influence of the ligand structure on immunochemical detection of the hapten. Histochemistry 84:169–178
Jesik CJ, Holland JM, Lee C (1982) An anatomic and histologic study of the rat prostate. The Prostate 3:81–97
Kendall CH, Roberts PA, Pringle JH, Lauder I (1991) The expression of parathyroid hormone messenger RNA in normal and abnormal parathyroid tissue. J Pathol 165:111–118
Komminoth P (1992) Digoxigenin as an alternative probe labeling for in situ hybridization. Diagn Mol Pathol 1:142–150
Lackie PM, Zuber C, Roth J (1991) Expression of polysialylated N-CAM during rat heart development. Differentiation 47:85–98
Langer PR, Waldrop AA, Ward DC (1981) Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc Natl Acad Sci USA 78:6633–6637
Larsson L-I, Hougaard DM (1992) Detection of gastrin and its messenger RNA in Zollinger-Ellison tumors by non-radioactive in situ hybridization and immunocytochemistry. Histochemistry 97:105–110
Long AA, Mueller J, Andre-Schwartz J, Barrett KJ, Schwartz R, Wolfe HJ (1992) High-specificity in situ hybridization: methods and application. Diagn Mol Pathol 1:45–57
Matusik RJ, Kreis C, McNicol P, Sweetland R, Mullin C, Fleming WH, Dodd JG (1986) Regulation of prostatic genes: role of androgens and zinc in gene expression. Biochem Cell Biol 64:601–607
Morimoto H, Monden T, Shimano T, Higashiyama M, Tamita N, Murotani M, Matsuura N, Okuda H, Mori T (1987) Use of sulfonated robes for in situ detection of amylase mRNA in formalin-fixed paraffin sections of human pancreas and submaxillary gland. Lab Invest 57:737–741
Morris RG, Arends MJ, Bishop PE, Sizer K, Duvall E, Bird CC (1990) Sensitivity of digoxigenin and biotin labelled probes for detection of human papillomavirus by in situ hybridization. J Clin Pathol 43:800–805
Nakane PK, Koji T, Izumi S (1989) In situ localization of mRNA with T-T dimerized DNA probes. Hum Cell 2:356–362
Niedobitek G, Finn T, Herbst H, Bornhöft G, Gerdes J, Stein H (1988) Detection of viral DNA by in situ hybridization using bromodeoxyuridine-labeled DNA probes. Am J Pathol 131:1–4
Price D (1963) Comparative aspects of development and structure in the prostate. In: Vollmer EP (ed) Biology of the prostate and related tissues. US Department of Health, Education and Welfare, Bethesda, Md, pp 1–27
Roth J (1989) Postembedding labeling on Lowicryl K4M tissue sections: detection and modification of cellular components. Methods Cell Biol 31:513–551
Roth J, Bendayan M, Orci L (1978) Ultrastructural localization of intracellular antigens by the use of protein A-gold complex. J Histochem Cytochem 26:1074–1081
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Sata T, Zuber C, Roth J (1990) Lectin-digoxigenin conjugates: a new hapten system for glycoconjugate cytochemistry. Histochemistry 94:1–11
Shan LX, Rodriguez MC, Janne OA (1990) Regulation of androgen receptor protein and mRNA concentration by androgens in rat ventral prostate and seminal vesicles and in human hepatoma cells. Mol Endocrinol 4:1636–1646
Shroyer KP, Nakane PKJ (1983) Use of DNP-labeled cDNA for in situ hybridization. J Cell Biol 97:377a
Takeda H, Chodak G, Mutchnik S, Nakamoto T, Chang C (1990) Immunohistochemical localization of androgen receptors with mono and polyclonal antibodies to androgen receptor. J Endocrinol 126:17–25
van den Brule AJC, Cromme FV, Snijders PJF, Smit L, Oudejans CBM, Baak JPA, Meijer CJLM, Waldboomers JMM (1991) Nonradioactive RNA in situ hybridization detection of human papillomavirus 16-E7 transcripts in squamous cell carcinomas of the uterine cervix using confocal laser scan microscopy. Am J Pathol 139:1037–1045
Venole P (1990) Digoxigenin-labeled probes can detect single-copy genes in human metaphase chromosomes. BioTechniques 9:200–204
Yu M, Leav BA, Leav I, Merk FB, Wolfe HJ, Ho S-M (1993) Early alterations in ras protooncogene mRNA expression in testosterone and estradiol-17β induced prostatic dysplasia of Noble rats. Lab Invest 68 (in press)
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Komminoth, P., Merk, F.B., Leav, I. et al. Comparison of 35S- and digoxigenin-labeled RNA and oligonucleotide probes for in situ hybridization. Histochemistry 98, 217–228 (1992). https://doi.org/10.1007/BF00271035
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DOI: https://doi.org/10.1007/BF00271035