Nucleic Acids Research Advance Access originally published online on July 17, 2007
Nucleic Acids Research 2007 35(15):e94; doi:10.1093/nar/gkm510
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Nucleic Acids Research, 2007, Vol. 35, No. 15 e94
© 2007 The Author(s)
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.
Methods Online |
Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)
1Department of Epidemiology and Population Health, 2Department of Pathology, 3Department of Molecular Genetics and 4Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA
*To whom correspondence should be addressed. Tel: +1 718 430 3430; Fax: +1 718 430 8653; Email: oloudig{at}aecom.yu.edu
Received January 19, 2007. Revised May 29, 2007. Accepted June 13, 2007.
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-dT24-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.