# Advanced Omics and Radiobiological Tissue Archives: The Future in the Past
## 3 Main Challenges with Omics Analysis of FFPE  
For decades FFPE tissues offered the standard materials for histopathological analysis due to the well-preserved morphological architecture, ease of sample preparation, and stability during long-term storage. The rapid development of emerging molecular technologies provides new opportunities for retrospective studies. For a long time, applying such advanced approaches as omics analysis to archival samples were considered an almost impossible task, mainly due to the loss of macromolecular integrity and quality during the rough fixation, delayed embedding and conditions of long-term storage. The sequence of events strongly affected the quality and quantity of protein and nucleic acids yielded from FFPE tissue and hampered the accurate qualitative and quantitative analysis [3,46]. The changes occurring during cross-linking of proteins during the fixation of FFPE tissue were investigated in several studies [47, 48, 49]. The most significant consequence of formaldehyde fixation is the generation of a methylol modification at free lysine residues that severely interferes with protein extraction and separation [47,50]. The modified lysine residues remain inaccessible to protease during digestion, a phenomenon that results in a preference for identifying tryptic peptides with the C-terminal arginine over lysine [47,50]. Furthermore, the use of conventional protein labelling techniques for quantitative proteomic analysis of FFPE tissues is even less effective [47,50]. Since the majority of the chemical labels used in quantitative proteomics target lysine residues, the formalin modification of lysin leads to inefficient labelling of FFPE material [51, 52, 53]. Several studies were conducted to evaluate different protocols for the extraction of proteins of FFPE tissues [8, 9, 10, 11]. DNA, as the most stable species among biological macromolecules, is subject to the cross-linking of cytosine residues, or spontaneous deamination/depurination of nucleotides and fragmentation during the fixation procedure [54,55]. The formalin fixation not only affects the quantity and purity of the DNA yield, but also causes misinterpretation of the DNA sequence, particularly due to an increase in the level of artifactual mutation of cytosine (C) to thymine (T) and guanine (G) to adenine (A) (C:G > T:A) [56]. Yet another known problem is the high fragmentation of DNA, probably caused by warm ischemic time in operating rooms, or the type of formalin (buffered versus unbuffered) used, time of fixation that all may result in low quantities DNA yields and therefore low quantities of amplifying templates for analysis. In addition, formalin fixation produces non-reproducible sequence artefacts due to DNA fragmentation and hydrolytic delamination [57]. Several studies have reported the protocols to overcome these limitations for DNA microarray and DNA sequencing [3; 56; 57; 58]. These protocols also include bioinformatics or statistical tools to allow for different mutation findings in fresh-frozen or FFPE tissue, considering different allelic fractions, or distinguishing between clonal and subclonal mutations. In the same line of evidence, mRNA extracted from FFPE tissues is mostly fragmented, degraded and modified. Formaldehyde modification of RNA interferes with the base-pairing necessary for hybridization and introduces the cross-linking of RNA to other macromolecules [58; 59]. However, recent studies have demonstrated the feasibility of RNA analysis approaches including RNA seq in FFPE samples [60; 61; 62; 63]. In contrast to mRNA, miRNA showed more stability and compatibility in FFPE for expression analysis [64; 65; 66; 67]. The short length of miRNA is probably responsible for the fact that they are minimally affected by methylol cross-links during the fixation procedure, therefore their expression profile is comparable to those from frozen tissue samples [67; 68]. The conditions of FFPE preparation also pose a challenge for other omics approaches such as metabolomics and lipidomics.