title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT).
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 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.
authors.affiliation: Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY, USA. oloudig@aecom.yu.edu
authors.forename: Olivier
authors.initials: O
authors.lastname: Loudig
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Milova
authors.forename: Margaret
authors.initials: M
authors.lastname: Brandwein-Gensler
authors.forename: Aldo
authors.initials: A
authors.lastname: Massimi
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Belbin
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Childs
authors.forename: Robert H
authors.initials: RH
authors.lastname: Singer
authors.forename: Thomas
authors.initials: T
authors.lastname: Rohan
authors.forename: Michael B
authors.initials: MB
authors.lastname: Prystowsky
mesh_terms: D018070:Biological Specimen Banks; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis; D005260:Female; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D020869:Gene Expression Profiling / Q000379:methods*; D052138:Genes, Neoplasm; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016612:Paraffin Embedding; D018075:RNA, Complementary / Q000096:biosynthesis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D048348:Reverse Transcription; D013698:Templates, Genetic
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D005404:Fixatives; D018075:RNA, Complementary; D005557:Formaldehyde