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Eisele, Sylvia (2013): Gen-Expressionsanalyse aus autoptischen Formalin-fixierten und Paraffin- eingebetteten Gewebeproben von Multiple Sklerose Patienten. Dissertation, LMU München: Faculty of Medicine



A protocol for RNA isolation from FFPE brain tissue was introduced and optimized in the laboratory. It was demonstrated that both, RNA yield and the ratio of light absorption at 260 nm vs. 280 nm (OD 260/280) in FFPE tissue are comparable to frozen tissue (23). A total of 27 archival brain specimens of 11 MS donors obtained from different brain banks were screened for the ability to amplify the housekeeping gene PPIA as well as miRNA 181a and miR 124. Results were compared to amplification of the same transcripts in 9 frozen MS tissue samples of 9 MS patients. The ability to amplify PPIA in FFPE tissue specimens was very heterogeneously distributed and the loss of amplifiable transcript copies ranged from 45 fold to 200 000 fold as compared to frozen tissue. In some archival samples PPIA could not be detected at all. These specimens were considered not suitable for further qPCR analysis. In contrast, the amplification ofmiRNA 181a and miR 124 in FFPE tissue was tremendously stable with an average loss of amplifiability of 1.7 fold only (23). Among several factors which possibly have an influence on impaired transcript amplification in FFPE tissue, the effect of length of formalin fixation was investigated in more detail. It was shown that duration of formalin fixation had great impact on loss of subsequent amplification of coding transcripts (e. g. PPIA). Compared to frozen tissue, PPIA amplification was reduced by ~15 fold in samples which were formalin-fixed for a day-long period, which is in contrast to a reduction of PPIA amplification by ~200 fold in specimens which had been fixed for years (23). Here again, miRNA amplification was demonstrated to be remarkably stable in the same FFPE tissue samples (23). Based on the stable miRNA detection in FFPE tissue specimens, 18 FFPE tissue specimens (MS n=13, healthy donor n=5) were included in a study which compared the miRNA expression pattern in MS lesions to healthy brain tissue by qPCR analysis of 365 mature miRNAs (42). Furthermore, an experimental setup was established which allows for precise dissection of MS lesions from surrounding normal appearing white matter (NAWM). To this end, FFPE sections were obtained using a microtome, were flattened in a DEPC water bath and mounted on PEN membrane coated slides. RNA yield and amplification of PPIA were not altered by this approach. Parallel tissue sections were stained with Luxol Fast Blue (LFB) and served as a model to help with the precise dissection of MS lesions. This setup was applied to 5 FFPE tissue samples (MS lesion n=3, healthy donor n=2). RNA was isolated from the dissected tissue specimens to analyse differential expression of 84 extracellular matrix (ECM) related genes in MS lesions compared to healthy tissue using TaqMan® Low Density Array qPCR technology. This was compared to a data set derived from frozen tissue samples that had been processed in a similar way. Detection of gene regulation (MS/healthy) in FFPE tissue was found to be reliable and comparable to frozen tissue, provided that the selected genes were of sufficient abundance (23). The up-regulation of the extracellular matrix component decorin could be validated on protein level by immuno-histochemistry in the same FFPE MS lesions. This result was published as part of a study which investigated the expression of several extracellular matrix related genes in MS lesions with frozen tissue, e.g. collagens and the protein biglycan (61). Furthermore this study showed that fibrillar collagens, biglycan and decorin are part of the perivascular fibrosis. These molecules are expressed inproximity to tissue invading immune cells, therefore suggesting a possible disease modifying function (61). In summary, this work presents a detailed protocol for the use of autoptic FFPE tissue specimens to obtain gene expression profiles from dissected MS lesions (23). This protocol was implemented as part of a study which investigated alterations of ECM in MS lesions (61) and contributed to obtain the first miRNA profile in MS lesions (42).