Gen-Expressionsanalyse aus autoptischen Formalin-fixierten und Paraffin- eingebetteten Gewebeproben von Multiple Sklerose Patienten

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