Novel genetic and epigenetic alterations in ... - Ous-research.no

DISCUSSIONFresh-frozen versus formalin embedded tissueIn the present study both fresh frozen and formalin-fixed paraffin embedded tissue sampleshave been used. Formalin fixation is an excellent way of preserving good histological detailsin tissue sections, and is for this purpose superior to frozen sections. On the other hand, thispreservation method is inferior to snap- or fresh frozen tissue for retaining high qualityDNA and RNA. One of the main hurdles is the fragmentation of DNA as well as theinhibitory effects on PCR efficiency. This means that one should aspire to design short PCRproducts,and carefully optimize the reactions. In the present thesis we only analyzed shortPCR products with fragment analysis in the formalin embedded series, and thereby thisproblem was in large overcome. However, we did experience a small decrease in the successrate between the mutation results obtained from the fresh frozen test series versus thosefrom the formalin-fixed validation series (99.9%; range 99.1-100%, and 92%; range 0-100%,respectively).Methodological considerationsDNA methylation analysesBisulfite treatmentBisulfite treatment has been around for a while[137], but it was not until the early 1990s thatthe method was used to map 5-methyl cytosine[138;139]. With this, Frommer and Clarkefound a way of converting the non-readable epigenetic information into readable geneticinformation. Under acidic pH and high bisulfite concentration, bisulfite treatment convertsunmethylated, but not methylated, cytosine into uracil in a highly specific manner[140]. Thesubsequent difference in nucleotide sequence can be exploited to determine methylationstatus by numerous PCR-based methods. Insufficient bisulfite conversion fails to convert allunmethylated cytosines to thymine, making it difficult to discriminate methylated fromunmethylated cytosines in downstream analyses. The conversion rate can be limited by43