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In situ reverse transcription: the magic of strength and anonymity

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    0345708 - UEM-P 2011 RIV GB eng J - Journal Article
    Ligasová, Anna - Koberna, Karel
    In situ reverse transcription: the magic of strength and anonymity.
    Nucleic Acids Research. Roč. 38, č. 16 (2010), e167-e178. ISSN 0305-1048
    R&D Projects: GA ČR GA204/09/0973; GA AV ČR KJB500390701; GA AV ČR KAN200520801
    Institutional research plan: CEZ:AV0Z50390512
    Keywords : nucleic acids * DNA-RNA
    Subject RIV: EA - Cell Biology
    Impact factor: 7.836, year: 2010

    In this study, we describe an approach that enables a highly specific, effective and fast detection of polyadenylated RNA sequences in situ at the light and electron microscopy levels. The method developed is based on the incorporation of 5-bromo-2’-deoxyuridine into the growing cDNAstrand by means of the reverse transcriptase. We have shown that unlike the previously used deoxyuridine tagged with biotin or digoxigenin,5-bromo-2‘-deoxyuridine is ‘invisible’ in the DNA–DNA duplex but easily detectable in theDNA–RNA duplex. We have also shown that the replacement of deoxythymidine by 5-bromo-2‘-deoxyuridine considerably stabilizes the growing DNA–RNA duplex, thus enabling the one-step detection of polyadenylated RNA in structurally well-preserved cells.
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