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 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.
Loudig
Olivier
O
Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY, USA. oloudig@aecom.yu.edu
Milova
Ekaterina
E
Brandwein-Gensler
Margaret
M
Massimi
Aldo
A
Belbin
Thomas J
TJ
Childs
Geoffrey
G
Singer
Robert H
RH
Rohan
Thomas
T
Prystowsky
Michael B
MB
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
D017931:DNA Primers; D018076:DNA, Complementary; D005404:Fixatives; D018075:RNA, Complementary; D005557:Formaldehyde