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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.

We have developed RNA expression microarrays (REMs), in which each spot on a glass support is composed of a population of cDNAs synthesized from a cell or tissue sample. We used simultaneous hybridization with test and reference (housekeeping) genes to calculate an expression ratio based on normalization with the endogenous reference gene. A test REM containing artificial mixtures of liver cDNA and dilutions of the bacterial LysA gene cDNA demonstrated the feasibility of detecting transcripts at a sensitivity of four copies of LysA mRNA per liver cell equivalent. Furthermore, LysA cDNA detection varied linearly across a standard curve that matched the sensitivity of quantitative real-time PCR. In REMs with real samples, we detected organ-specific expression of albumin, Hnf-4 and Igfbp-1, in a set of mouse organ cDNA populations and c-Myc expression in tumor samples in paired tumor/normal tissue cDNA samples. REMs extend the use of classic microarrays in that a single REM can contain cDNAs from hundreds to thousands of cell or tissue samples each representing a specific physiological or pathophysiological state. REMs will extend the analysis of valuable samples by providing a common broad based platform for their analysis and will promote research aimed at defining gene functions, by broadening our understanding of their expression patterns in health and disease.

Testicular germ cell tumors (TGCTs) show, for the most part, a characteristic cytogenetic aberration carrying one or more copies of i(12p). Mostly i(12p)-negative TGCTs also show an overrepresentation of 12p11p12 sequences. Critical genes in the amplicon defined by 12p11p12 may be involved in the progression of TGCTs and other tumors. To identify directly the expressed sequence tags (ESTs) involved in the amplicon, we performed comparative genomic hybridization array analysis of five TGCTs demonstrated to exhibit high DNA copy number in this region. The use of arrays spotted with 8,060 IMAGE complementary DNA clones amplified by means of the polymerase chain reaction (PCR) identified 18 amplified ESTs. Most of them were mapped to 12p and the remainder have been mapped to 12. We also identified new testis-associated transcripts by PCR with reverse transcription of the amplified EST sequences. Using combined RNA-array and real-time-beacon PCR analysis, we identified two amplified and overexpressed ESTs. The level of expression in these ESTs was four- to sixfold higher in one tumor sample compared with normal testis. The expression ratios obtained by real-time-beacon PCR analysis correlated with those obtained by RNA-array data analysis, thus validating the techniques used. However, we failed to find an amplified and overexpressed EST commonly overexpressed in all the tumor samples tested, perhaps because the arrays used might only represent approximately 10% of human genes. Therefore we decided to design a 12p array containing 768 ESTs, representing all the 12p-mapped ESTs found in the available databases. Results of comparative genomic hybridization and RNA-array analysis of TGCTs using the 12p array will be presented.

cDNA microarray technology provides a novel approach to identify individual target genes and survey global genetic programs under the control of specific signalling pathways in mammalian systems. Smad2 and Smad3 are highly homologous members of the receptor-regulated subfamily of Smad proteins with a central role in TGF- signalling and target gene regulation.