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In situ reverse transcription: the magic of strength and anonymity

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 cDNA strand by means...

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Detalles Bibliográficos
Autores principales: Ligasová, Anna, Koberna, Karel
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2938209/
https://www.ncbi.nlm.nih.gov/pubmed/20627869
http://dx.doi.org/10.1093/nar/gkq619
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author Ligasová, Anna
Koberna, Karel
author_facet Ligasová, Anna
Koberna, Karel
author_sort Ligasová, Anna
collection PubMed
description 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 cDNA strand 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 the DNA–RNA duplex. This feature is an important pre-requisite for the correct interpretation of the data obtained, as our results strongly indicate that reverse transcriptase uses DNA breaks as primers efficiently. 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. The method developed provides a highly specific signal with the signal/noise ratio higher than 130 for permeabilized cells and 25 for conventional acrylic resin sections under the conditions used. When the high pressure freezing technique followed by the freeze substitution is employed for the cell's preparation, the ratio is higher than 80.
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spelling pubmed-29382092010-09-13 In situ reverse transcription: the magic of strength and anonymity Ligasová, Anna Koberna, Karel Nucleic Acids Res Methods Online 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 cDNA strand 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 the DNA–RNA duplex. This feature is an important pre-requisite for the correct interpretation of the data obtained, as our results strongly indicate that reverse transcriptase uses DNA breaks as primers efficiently. 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. The method developed provides a highly specific signal with the signal/noise ratio higher than 130 for permeabilized cells and 25 for conventional acrylic resin sections under the conditions used. When the high pressure freezing technique followed by the freeze substitution is employed for the cell's preparation, the ratio is higher than 80. Oxford University Press 2010-09 2010-07-13 /pmc/articles/PMC2938209/ /pubmed/20627869 http://dx.doi.org/10.1093/nar/gkq619 Text en © The Author(s) 2010. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/2.5 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Ligasová, Anna
Koberna, Karel
In situ reverse transcription: the magic of strength and anonymity
title In situ reverse transcription: the magic of strength and anonymity
title_full In situ reverse transcription: the magic of strength and anonymity
title_fullStr In situ reverse transcription: the magic of strength and anonymity
title_full_unstemmed In situ reverse transcription: the magic of strength and anonymity
title_short In situ reverse transcription: the magic of strength and anonymity
title_sort in situ reverse transcription: the magic of strength and anonymity
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2938209/
https://www.ncbi.nlm.nih.gov/pubmed/20627869
http://dx.doi.org/10.1093/nar/gkq619
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