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Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)
Gene expression profiling of formalin-fixed and paraffin-embedded (FFPE) specimens, banked from completed clinical trials and routine clinical care, has the potential to yield valuable information implicating and linking genes with clinical parameters. In order to prepare high-quality cDNA from high...
Autores principales: | , , , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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Oxford University Press
2007
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1976468/ https://www.ncbi.nlm.nih.gov/pubmed/17636051 http://dx.doi.org/10.1093/nar/gkm510 |
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author | Loudig, Olivier Milova, Ekaterina Brandwein-Gensler, Margaret Massimi, Aldo Belbin, Thomas J. Childs, Geoffrey Singer, Robert H. Rohan, Thomas Prystowsky, Michael B. |
author_facet | Loudig, Olivier Milova, Ekaterina Brandwein-Gensler, Margaret Massimi, Aldo Belbin, Thomas J. Childs, Geoffrey Singer, Robert H. Rohan, Thomas Prystowsky, Michael B. |
author_sort | Loudig, Olivier |
collection | PubMed |
description | Gene expression profiling of formalin-fixed and paraffin-embedded (FFPE) specimens, banked from completed clinical trials and routine clinical care, has the potential to yield valuable information implicating and linking genes with clinical parameters. In order to prepare high-quality cDNA from highly fragmented FFPE-RNA, previously precluded from high-throughput analyses, we have designed a novel strategy based on the nucleic acid restoration of incomplete cDNA sequences prior to T7 in vitro transcription (IVT) amplification. We describe this strategy as complementary-template reverse-transcription (CT-RT) because short single-stranded T7-oligo-dT(24)-VN-DNA sequences, obtained from FFPE-RNA, are used as primers for the RT of complementary RNA templates contained in a sense-RNA library. We validated our assay by determining the correlation between expression profiles of a matched 10-year-old frozen and FFPE breast cancer sample. We show that T7 IVT-amplification of cDNA transcripts restored by CT-RT is a specific and reliable process that allows recovery of transcriptional features undetectable by direct T7 IVT-amplification of FFPE-RNA. Furthermore, CT-RT restored 35–41% of the transcripts from archived breast and cervical specimens when compared to matched frozen tissue; and profiles included tissue-specific transcripts. Our results indicate that CT-RT allows microarray profiling of severely degraded RNA that could not be analyzed by previous methods. |
format | Text |
id | pubmed-1976468 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2007 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-19764682007-09-26 Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT) Loudig, Olivier Milova, Ekaterina Brandwein-Gensler, Margaret Massimi, Aldo Belbin, Thomas J. Childs, Geoffrey Singer, Robert H. Rohan, Thomas Prystowsky, Michael B. Nucleic Acids Res Methods Online Gene expression profiling of formalin-fixed and paraffin-embedded (FFPE) specimens, banked from completed clinical trials and routine clinical care, has the potential to yield valuable information implicating and linking genes with clinical parameters. In order to prepare high-quality cDNA from highly fragmented FFPE-RNA, previously precluded from high-throughput analyses, we have designed a novel strategy based on the nucleic acid restoration of incomplete cDNA sequences prior to T7 in vitro transcription (IVT) amplification. We describe this strategy as complementary-template reverse-transcription (CT-RT) because short single-stranded T7-oligo-dT(24)-VN-DNA sequences, obtained from FFPE-RNA, are used as primers for the RT of complementary RNA templates contained in a sense-RNA library. We validated our assay by determining the correlation between expression profiles of a matched 10-year-old frozen and FFPE breast cancer sample. We show that T7 IVT-amplification of cDNA transcripts restored by CT-RT is a specific and reliable process that allows recovery of transcriptional features undetectable by direct T7 IVT-amplification of FFPE-RNA. Furthermore, CT-RT restored 35–41% of the transcripts from archived breast and cervical specimens when compared to matched frozen tissue; and profiles included tissue-specific transcripts. Our results indicate that CT-RT allows microarray profiling of severely degraded RNA that could not be analyzed by previous methods. Oxford University Press 2007-08 2007-07-17 /pmc/articles/PMC1976468/ /pubmed/17636051 http://dx.doi.org/10.1093/nar/gkm510 Text en © 2007 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Loudig, Olivier Milova, Ekaterina Brandwein-Gensler, Margaret Massimi, Aldo Belbin, Thomas J. Childs, Geoffrey Singer, Robert H. Rohan, Thomas Prystowsky, Michael B. Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT) |
title | Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT) |
title_full | Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT) |
title_fullStr | Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT) |
title_full_unstemmed | Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT) |
title_short | Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT) |
title_sort | molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (ct-rt) |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1976468/ https://www.ncbi.nlm.nih.gov/pubmed/17636051 http://dx.doi.org/10.1093/nar/gkm510 |
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