Genome-wide expression assay comparison across frozen and fixed postmortem brain tissue samples

<p>Abstract</p> <p>Background</p> <p>Gene expression assays have been shown to yield high quality genome-wide data from partially degraded RNA samples. However, these methods have not yet been applied to postmortem human brain tissue, despite their potential to overcome poor RNA quality and other technical limitations inherent in many assays. We compared cDNA-mediated annealing, selection, and ligation (DASL)- and <it>in vitro </it>transcription (IVT)-based genome-wide expression profiling assays on RNA samples from artificially degraded reference pools, frozen brain tissue, and formalin-fixed brain tissue.</p> <p>Results</p> <p>The DASL-based platform produced expression results of greater reliability than the IVT-based platform in artificially degraded reference brain RNA and RNA from frozen tissue-based samples. Although data associated with a small sample of formalin-fixed RNA samples were poor when obtained from both assays, the DASL-based platform exhibited greater reliability in a subset of probes and samples.</p> <p>Conclusions</p> <p>Our results suggest that the DASL-based gene expression-profiling platform may confer some advantages on mRNA assays of the brain over traditional IVT-based methods. We ultimately consider the implications of these results on investigations of neuropsychiatric disorders.</p

Clinical relevance of DNA microarray analyses using archival formalin-fixed paraffin-embedded breast cancer specimens

Abstract Background The ability of gene profiling to predict treatment response and prognosis in breast cancers has been demonstrated in many studies using DNA microarray analyses on RNA from fresh frozen tumor specimens. In certain clinical and research situations, performing such analyses on archival formalin fixed paraffin-embedded (FFPE) surgical specimens would be advantageous as large libraries of such specimens with long-term follow-up data are widely available. However, FFPE tissue processing can cause fragmentation and chemical modifications of the RNA. A number of recent technical advances have been reported to overcome these issues. Our current study evaluates whether or not the technology is ready for clinical applications. Methods A modified RNA extraction method and a recent DNA microarray technique, cDNA-mediated annealing, selection, extension and ligation (DASL, Illumina Inc) were evaluated. The gene profiles generated from FFPE specimens were compared to those obtained from paired fresh fine needle aspiration biopsies (FNAB) of 25 breast cancers of different clinical subtypes (based on ER and Her2/neu status). Selected RNA levels were validated using RT-qPCR, and two public databases were used to demonstrate the prognostic significance of the gene profiles generated from FFPE specimens. Results Compared to FNAB, RNA isolated from FFPE samples was relatively more degraded, nonetheless, over 80% of the RNA samples were deemed suitable for subsequent DASL assay. Despite a higher noise level, a set of genes from FFPE specimens correlated very well with the gene profiles obtained from FNAB, and could differentiate breast cancer subtypes. Expression levels of these genes were validated using RT-qPCR. Finally, for the first time we correlated gene expression profiles from FFPE samples to survival using two independent microarray databases. Specifically, over-expression of ANLN and KIF2C, and under-expression of MAPT strongly correlated with poor outcomes in breast cancer patients. Conclusion We demonstrated that FFPE specimens retained important prognostic information that could be identified using a recent gene profiling technology. Our study supports the use of FFPE specimens for the development and refinement of prognostic gene signatures for breast cancer. Clinical applications of such prognostic gene profiles await future large-scale validation studies