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In situ reverse transcription: the magic of strength and anonymity
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SYSNO ASEP 0345708 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title In situ reverse transcription: the magic of strength and anonymity Author(s) Ligasová, Anna (UEM-P)
Koberna, Karel (UEM-P)Source Title Nucleic Acids Research. - : Oxford University Press - ISSN 0305-1048
Roč. 38, č. 16 (2010), e167-e178Number of pages 12 s. Language eng - English Country GB - United Kingdom Keywords nucleic acids ; DNA-RNA Subject RIV EA - Cell Biology R&D Projects GA204/09/0973 GA ČR - Czech Science Foundation (CSF) KJB500390701 GA AV ČR - Academy of Sciences of the Czech Republic (AV ČR) KAN200520801 GA AV ČR - Academy of Sciences of the Czech Republic (AV ČR) CEZ AV0Z50390512 - UEM-P (2005-2011) UT WOS 000281720500007 DOI 10.1093/nar/gkq619 Annotation 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. Workplace Institute of Experimental Medicine Contact Lenka Koželská, lenka.kozelska@iem.cas.cz, Tel.: 241 062 218, 296 442 218 Year of Publishing 2011
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