An in situ hybridization protocol to detect rare mRNA expressed in neural tissue using biotin-labelled oligonucleotide probes.
The use of the non-radioactive in situ hybridization protocols has allowed in general to obtain a better resolution of different transcripts at histological and cytological levels with a shortening of the developmental time. The common protocols using digoxigenin and biotin-labelled probes share a considerable limitation depending on the amount of the transcripts present in the tissues. This problem becomes more evident when oligonucleotide probes are used, because of their small size and lower ability to give sufficient signal amplification. The protocol reported here allows to localize rare mRNA expressed in a tissue, using a combination of two biotin-labelled oligonucleotide probes followed by streptavidin-peroxidase and biotinyl tyramide amplification system.
Dipartimento di Biologia Cellulare e dello Sviluppo, Università La Sapienza, P.le Aldo Moro, 5, 00185 Rome, Italy. tata@axcasp.caspur.it
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Tata
D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives; D017403:In Situ Hybridization / Q000379:methods*; D009475:Neurons, Afferent / Q000502:physiology; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification*; D051381:Rats; D017208:Rats, Wistar; D043587:Receptor, Muscarinic M3; D043588:Receptor, Muscarinic M4; D011976:Receptors, Muscarinic / Q000235:genetics; D014439:Tyramine / Q000031:analogs & derivatives
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D043587:Receptor, Muscarinic M3; D043588:Receptor, Muscarinic M4; D011976:Receptors, Muscarinic; C111551:biotinyltyramide; D001710:Biotin; D014439:Tyramine