Abstract
In situ hybridization experiments are stringently dependent on the quality of the probes, which should be single-stranded when efficient comparison of signals obtained with antisense and control sense probes are needed. In this report, we describe an optimized synthesis of radioactive single-stranded DNA probes, without vector cloning and requiring a unique polymerization step. The sequence region selected as probe is amplified by polymerase chain reaction in the presence of radiolabelled nucleotides. The sense and antisense probes are then yielded by the action of the λ bacteriophage exonuclease, which can specifically eliminate one out of the two strands of the amplified fragments. In this way, sense and antisense probes with identical length and specific activity can be generated by selecting the primer to be phosphorylated. We have verified the efficiency of our probes for in situ hybridization of the clusterin transcripts within the peripheral olfactory system, after surgical lesion of its synaptic target.
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References
Barber, P.C. & Lindsay, R.M. (1982) Schwann cells of the olfactory nerves contain glial fibrillary acidic protein and resemble astrocytes. Neuroscience 7, 3077-90.
Bean, A.J., Simons, J.F. & HÖkfelt, T. (1993) Production of labeled cRNA probes without vector cloning: application to localization of basic fibroblast growth factor and tyrosine hydroxylase mRNAs by in situ hybridization histochemistry. Mol. Cell. Neurosci. 4, 216-21.
Buttyan, R., Olsson, C.A., Pintar, J., Chang, C., Bandyk, M., Ng, P.-Y. & Sawczuk, I.S. (1989) Induction of the TRPM-2 gene in cells undergoing programmed cell death. Mol. Cell. Biol. 9, 3473-81.
Cox, K.H., DeLeon, D.V., Angerer, L.M. & Angerer, R.C. (1984) Detection of mRNAs in sea urchin embryos by in situ hybridization using assymetric RNA probes. Dev. Biol. 101, 485-502.
Danik, M., Chabot, J.-G, Hassan-Gonzalez, D., Suh, M. & Quirion, R. (1993) Localization of sulfated glycoprotein-2/clusterin mRNA in the rat brain by in situ hybridization. J. Comp. Neurol. 334, 209-27.
Doucette, R. (1990) Glial influences on axonal growth in the primary olfactory system. Glia 3, 433-49.
Dragunow, M., Preston, K., Dodd, J., Young, D., Lawlor, P. & Christie, D. (1995) Clusterin accumulates in dying neurons following status epilepticus. Mol. Brain Res. 32, 279-90.
Duguid, J.R., Bohmont, C.W., Lui, C.W. & Tourte-Lotte, W.W. (1989) Changes in brain gene expression shared by scrapie and Alzheimer disease. Proc. Natl. Acad. Sci. USA 86, 7260-4.
Guenette, R.S., Corbeil, H.B., LÉger, J., Wong, K., MÉzl, V., Mooibroek, M. & Tenniswod, M. (1994) Induction of gene expression during involution of the lactating mammary gland of the rat. J. Mol. Endocrinol. 12, 47-60.
Hannon, K., Johnstone, E., Craft, L.S., Little, S.P., Smith II, C.K., Heiman, M.L. & Santerre, R.F. (1993) Synthesis of PCR-derived, single-stranded DNA probes suitable for in situ hybridization. Anal. Biochem. 212, 421-7.
Higuchi, R. & Ochman, H. (1989) Production of single-stranded DNA templates by exonuclease digestion following the polymerase chain reaction. Nucleic Acids Res. 17, 5865.
HÖfler, H. (1990) Principles of in situ hybridization. In In situ hybridization (edited by Polak, J.M. & McGee, J.O.), pp. 15-29. Oxford: Oxford University Press.
Hsieh-Li, H.M., Witte, D.P., Weinstein, M., Branford, M., Li, H., Small, K. & Potter, S.S. (1995) Hoxa 11 structure, extensive antisense transcription, and function in male and female fertility. Development 121, 1373-85.
Logel, J., Dill, D. & Leonard, S. (1992) Synthesis of cRNA probes from PCR-generated DNA. Biotechniques 13, 604-10.
Lund, L.R., Romer, J., Thomasset, N., Solberg, H., Pyke, C., Bissel, M.J., Dano, K. & Werb, Z. (1996) Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and-dependent pathways. Development 122, 181-93.
McGeer, P.L., Kawamata, T. & Walker, D.G. (1992) Distribution of clusterin in Alzheimer brain tissue. Brain Res. 579, 337-41.
May, P.C. & Finch, C.E. (1992) Sulfated glycoprotein 2: new relationships of this multifunctional protein to neurodegeneration. Trends Neurosci. 15, 391-6.
May, P.C., Lampert-Etchells, M., Johnson, S.A., Poirier, J., Masters, J.N. & Finch, C.E. (1990) Dynamics of gene expression for hippocampal glycoprotein elevated in Alzheimer's disease and in response to experimental lesions in rat. Neuron 5, 831-9.
Mertz, L.M. & Rashtchian, A. (1994) Nucleotide imbalance and polymerase chain reaction: effects on DNA amplification and synthesis of high specific activity radioalabeled DNA probes. Anal. Biochem. 221, 160-5.
Michel, D., Chabot, J.-G., Moyse, E., Danik, M. & Quirion, R. (1992) Possible functions of a new genetic marker in central nervous system: the sulfated glycoprotein-2 (SGP-2). Synapse 11, 105-11.
Michel, D., Moyse, E., Brun, G. & Jourdan, F. (1994a) Induction of apoptosis in rat olfactory neuroepithelium. Neuroreport 5, 1329-32.
Michel, D., Moyse, E., Trembleau, A., Jourdan, F. & Brun, G. (1994b) Clusterin expression and associated early molecular events induced in olfactory sensory mucosa by synaptic target ablation (abstract). Second Clusterin Workshop. September 3–5, Coeur d'Alene, Idaho. p. 38.
Monti-Graziadei, G.A. & Graziadei, P.P.C. (1979) Neurogenesis and neuron regeneration in the olfactory system of mammals. II. Degeneration and reconstitution of the olfactory sensory neurons after axotomy. J. Neurocytol. 8, 197-213.
Norman, D.J., Feng, L., Cheng, S.S., Gubbay, J., Chan, E. & Heintz, N. (1995) The lurcher gene induces apoptotic death in cerebellar Purkinje cells. Development 121, 1183-93.
Pasinetti, G.M., Cheng, H.W., Moran, D.G., Lampertetchells, M., McNeill, T.H. & Finch, C.E. (1993) Astrocytic messenger RNA responses to striatal deafferentation in male rat. Neuroscience 53, 199-211.
Pasinetti, G.M., Johnson, S.A., Oda, T., Rozovski, I. & Finch, C.E. (1994) Clusterin (SGP-2) — a multifunctional glycoprotein with regional expression in astrocytes and neurons of the adult rat brain. J. Comp. Neurol. 339, 387-400.
Simmons, D.M., Arriza, J.L. & Swanson, L.W. (1989) A complete protocol for in situ hybridization of messenger RNAs in brain and other tissues with radio-labeled single-stranded probes. J. Histotechnol. 12, 169-81.
Yasuhara, O., Aimi, Y., Yamda, T., Matsuo, A., McGeeg, E.G. & McGeer, P.L. (1994) Clusterin as a marker for ischaemic Purkinje cells in human brain. Neurodegeneration 3, 325-9.
Zoli, M., Ferraguti, F., Zini, I., Bettuzzi, S. & Agnati, L.F. (1993) Increases in sulphated glycoprotein-2 messenger RNA levels in the rat brain after transient forebrain ischemia or partial mesodiencephalic hemitransection. Mol. Brain Res. 18, 163-77.
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Michel, D., Trembleau, A., Moyse, E. et al. Optimization of PCR/λ exonuclease-mediated synthesis of sense and antisense DNA probes for in situ hybridization. J Mol Hist 29, 685–693 (1997). https://doi.org/10.1023/A:1026456816464
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DOI: https://doi.org/10.1023/A:1026456816464