Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)

Olivier Loudig, Ekaterina Milova, Margaret Brandwein-Gensler, Aldo B. Massimi, Thomas J. Belbin, Geoffrey J. Childs, Robert H. Singer, Thomas E. Rohan, Michael B. Prystowsky

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

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 informationimplicating 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 reversetranscription (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 IVTamplification 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.

Original languageEnglish (US)
Article numbere94
JournalNucleic Acids Research
Volume35
Issue number15
DOIs
StatePublished - Aug 2007

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Paraffin
Formaldehyde
Reverse Transcription
RNA
Complementary DNA
Complementary RNA
Gene Expression Profiling
Nucleic Acids
Breast
Clinical Trials
Breast Neoplasms
Genes
In Vitro Techniques

ASJC Scopus subject areas

  • Genetics

Cite this

Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT). / Loudig, Olivier; Milova, Ekaterina; Brandwein-Gensler, Margaret; Massimi, Aldo B.; Belbin, Thomas J.; Childs, Geoffrey J.; Singer, Robert H.; Rohan, Thomas E.; Prystowsky, Michael B.

In: Nucleic Acids Research, Vol. 35, No. 15, e94, 08.2007.

Research output: Contribution to journalArticle

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AU - Belbin, Thomas J.

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