Efficacy of a multimolecular extraction from formalin-fixed paraffin-embedded tissue
Master of Science
Formalin-fixed paraffin-embedded tissues
Genetics and gene expression
DNA & RNA extraction
Genetic profiling variation
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As the genetic basis and gene expression components are being discovered for an increasing number of diseases and disorders there is a rising interest in using multiomic approaches to better understand these conditions. The large tissue banks of formalin fixed (FF) and formalin-fixed paraffin-embedded (FFPE) tissues present a unique opportunity to study rare conditions and difficult to sample organs such as brains. Current molecular studies on FFPE look at single molecule extractions, which provide only a partial picture of disease mechanisms. This study compared the efficacy of TRIzol®, a tri-molecule extraction method, to that of the traditional single molecule methods for DNA and RNA extraction in frozen, FF, and FFPE brain tissue using animal models. Optimization of this would allow for DNA and RNA to be obtained from the same sample providing a more accurate and comprehensive picture of the mechanism of disease or disorder. The quantity and purity of the DNA and RNA extracted from frozen, FF, and FFPE brain samples (aged for 3 years) were determined by spectrophotometry. Quality was determined by real-time PCR (qPCR) and reverse transcriptase qPCR (RT-qPCR) using beta-2-microglobulin (98bp) and glyceraldehyde 3-phosphate dehydrogenase (227bp) targets. Our study found no significant difference in efficacy between TRIzol® and the traditional methods employed in the literature, indicating that TRIzol® is comparable to the currently used, single molecule extractions when applied to FF and FFPE brain tissue. This finding requires further investigation with a larger set of samples, but if supported may help maximize the amount of information obtained from rare samples as multiple data sets would be obtained from a single sample. Unexpectedly, this study also observed a higher rate of success in extracting high quality RNA compared to DNA regardless of extraction method. Due to the low number of samples, the statistical significance of this observation could not be established however we feel that it warrants further investigation.