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Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization
Clinical tissues are prepared for histological analysis and long-term storage via formalin fixation and paraffin embedding (FFPE). The FFPE process results in fragmentation and chemical modification of RNA, rendering it less suitable for analysis by techniques that rely on reverse transcription (RT)...
Autores principales: | , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737328/ https://www.ncbi.nlm.nih.gov/pubmed/28854731 http://dx.doi.org/10.1093/nar/gkx471 |
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author | Credle, Joel J. Itoh, Christopher Y. Yuan, Tiezheng Sharma, Rajni Scott, Erick R. Workman, Rachael E. Fan, Yunfan Housseau, Franck Llosa, Nicolas J. Bell, W. Robert. Miller, Heather Zhang, Sean X. Timp, Winston Larman, H. Benjamin |
author_facet | Credle, Joel J. Itoh, Christopher Y. Yuan, Tiezheng Sharma, Rajni Scott, Erick R. Workman, Rachael E. Fan, Yunfan Housseau, Franck Llosa, Nicolas J. Bell, W. Robert. Miller, Heather Zhang, Sean X. Timp, Winston Larman, H. Benjamin |
author_sort | Credle, Joel J. |
collection | PubMed |
description | Clinical tissues are prepared for histological analysis and long-term storage via formalin fixation and paraffin embedding (FFPE). The FFPE process results in fragmentation and chemical modification of RNA, rendering it less suitable for analysis by techniques that rely on reverse transcription (RT) such as RT-qPCR and RNA-Seq. Here we describe a broadly applicable technique called ‘Ligation in situ Hybridization’ (‘LISH’), which is an alternative methodology for the analysis of FFPE RNA. LISH utilizes the T4 RNA Ligase 2 to efficiently join adjacent chimeric RNA–DNA probe pairs hybridized in situ on fixed RNA target sequences. Subsequent treatment with RNase H releases RNA-templated ligation products into solution for downstream analysis. We demonstrate several unique advantages of LISH-based assays using patient-derived FFPE tissue. These include >100-plex capability, compatibility with common histochemical stains and suitability for analysis of decade-old materials and exceedingly small microdissected tissue fragments. High-throughput DNA sequencing modalities, including single molecule sequencing, can be used to analyze ligation products from complex panels of LISH probes (‘LISH-seq’), which can be amplified efficiently and with negligible bias. LISH analysis of FFPE RNA is a novel methodology with broad applications that range from multiplexed gene expression analysis to the sensitive detection of infectious organisms. |
format | Online Article Text |
id | pubmed-5737328 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-57373282018-01-08 Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization Credle, Joel J. Itoh, Christopher Y. Yuan, Tiezheng Sharma, Rajni Scott, Erick R. Workman, Rachael E. Fan, Yunfan Housseau, Franck Llosa, Nicolas J. Bell, W. Robert. Miller, Heather Zhang, Sean X. Timp, Winston Larman, H. Benjamin Nucleic Acids Res Methods Online Clinical tissues are prepared for histological analysis and long-term storage via formalin fixation and paraffin embedding (FFPE). The FFPE process results in fragmentation and chemical modification of RNA, rendering it less suitable for analysis by techniques that rely on reverse transcription (RT) such as RT-qPCR and RNA-Seq. Here we describe a broadly applicable technique called ‘Ligation in situ Hybridization’ (‘LISH’), which is an alternative methodology for the analysis of FFPE RNA. LISH utilizes the T4 RNA Ligase 2 to efficiently join adjacent chimeric RNA–DNA probe pairs hybridized in situ on fixed RNA target sequences. Subsequent treatment with RNase H releases RNA-templated ligation products into solution for downstream analysis. We demonstrate several unique advantages of LISH-based assays using patient-derived FFPE tissue. These include >100-plex capability, compatibility with common histochemical stains and suitability for analysis of decade-old materials and exceedingly small microdissected tissue fragments. High-throughput DNA sequencing modalities, including single molecule sequencing, can be used to analyze ligation products from complex panels of LISH probes (‘LISH-seq’), which can be amplified efficiently and with negligible bias. LISH analysis of FFPE RNA is a novel methodology with broad applications that range from multiplexed gene expression analysis to the sensitive detection of infectious organisms. Oxford University Press 2017-08-21 2017-06-19 /pmc/articles/PMC5737328/ /pubmed/28854731 http://dx.doi.org/10.1093/nar/gkx471 Text en © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Methods Online Credle, Joel J. Itoh, Christopher Y. Yuan, Tiezheng Sharma, Rajni Scott, Erick R. Workman, Rachael E. Fan, Yunfan Housseau, Franck Llosa, Nicolas J. Bell, W. Robert. Miller, Heather Zhang, Sean X. Timp, Winston Larman, H. Benjamin Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization |
title | Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization |
title_full | Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization |
title_fullStr | Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization |
title_full_unstemmed | Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization |
title_short | Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization |
title_sort | multiplexed analysis of fixed tissue rna using ligation in situ hybridization |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737328/ https://www.ncbi.nlm.nih.gov/pubmed/28854731 http://dx.doi.org/10.1093/nar/gkx471 |
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