**2. Factors affecting nucleic acids extraction from FFPE tissues**

Difficulties in obtaining quality nucleic acids, especially RNA, cause degradation and chemi‐ cal modification, despite the fixation in formalin and the routine histological processing techniques used to preserve cellular morphology and tissues' protein integrity [3–4].

Nucleic acids extraction from FFPE tissue shows some critical issues that may affect the quality of the obtained DNA or RNA as well as these samples' subsequent amplification process. Such critical matters include tissue fixing and clamping as well as the post-fixing stage (tissue cutting preparations, deparaffinization and hydration processes, in addition to other stages of the extraction process itself, such as digestion and purification), which will be described below [9].

Pre-fixation is defined as the period between tissue collection and the beginning of the set‐ ting process. The material starts degrading shortly after its collection, right when the tissue is exposed to hypoxia and to the DNases and RNases found in the environment. The first biochemical modifications emerge after 10 min of anoxia. Thus, it is very important to re‐ duce the pre-clamping time to seconds [4, 9].

The fixing conditions (time, temperature and fixative type) and, in some cases, the descaling processes alter material preservation and directly influence the quantity and quality of the obtained nucleic acid. Fixations kept in formalin solution for more than a week destroy the nucleic acids and lead to the cross-linking of all tissue components. It results in highly frag‐ mented nucleic acids, which are more resistant to the extraction process [2, 3, 9].

Chemical studies have shown that formaldehyde breaks hydrogen bonds in double-strand‐ ed DNA adenine- and thymine-rich regions. It creates new chemical interactions in protein folding, thus resulting in bonds between DNA proteins and DNA fragmentation [10].

nucleic acids with the same quality, especially RNA, since they degrade and chemically

Formalin replacement by other tissue fixatives such as Bouin, Carnoy, alcohol or HOPE (glu‐ tamic acid buffer Hepes-mediated organic solvent effect protection) may be an alternative to reverse the problem [4, 5]. However, since formalin is the fixative of choice in most patholo‐ gy departments, several research groups have sought to reverse the chemical changes

Although several papers describe techniques that allow FFPE tissue nucleic acid extracting process [6–8, 3, 1, 4], so far there is no consensus in the literature about the best protocol to

The studies based on such approach not often detail the used methodology. Besides, not all the published techniques were reproduced by our group. It took us approximately 8 months

The current chapter aims to describe the factors affecting the process of nucleic acid extrac‐ tion from FFPE tissue, compare the available protocols and to describe the modifications de‐ veloped by our group in some protocols that enable nucleic acids obtainment in satisfactory

Difficulties in obtaining quality nucleic acids, especially RNA, cause degradation and chemi‐ cal modification, despite the fixation in formalin and the routine histological processing

Nucleic acids extraction from FFPE tissue shows some critical issues that may affect the quality of the obtained DNA or RNA as well as these samples' subsequent amplification process. Such critical matters include tissue fixing and clamping as well as the post-fixing stage (tissue cutting preparations, deparaffinization and hydration processes, in addition to other stages of the extraction process itself, such as digestion and purification), which will

Pre-fixation is defined as the period between tissue collection and the beginning of the set‐ ting process. The material starts degrading shortly after its collection, right when the tissue is exposed to hypoxia and to the DNases and RNases found in the environment. The first biochemical modifications emerge after 10 min of anoxia. Thus, it is very important to re‐

The fixing conditions (time, temperature and fixative type) and, in some cases, the descaling processes alter material preservation and directly influence the quantity and quality of the obtained nucleic acid. Fixations kept in formalin solution for more than a week destroy the nucleic acids and lead to the cross-linking of all tissue components. It results in highly frag‐

mented nucleic acids, which are more resistant to the extraction process [2, 3, 9].

techniques used to preserve cellular morphology and tissues' protein integrity [3–4].

to standardize the DNA and RNA extraction process in our FFPE tissues research.

**2. Factors affecting nucleic acids extraction from FFPE tissues**

modify such acids [3, 4].

28 Nucleic Acids - From Basic Aspects to Laboratory Tools

caused by this fixative type.

be used in this type of material.

be described below [9].

duce the pre-clamping time to seconds [4, 9].

quantity and quality for molecular biology studies.

RNA messenger (mRNA) obtained from FFPE tissues is often not intact. It is usually degrad‐ ed to less than 300 base pairs [1]. However, Hamatani et al. (2006) [11] found that 80% of the RNA samples presenting 60 base pairs may be sufficiently amplified by polymerase chain reactions (PCR). All post-fixation stages, such as the paraffin blocks attainment sections, are also essential to the obtainment of high-quality nucleic acids.

Contamination is one of the critical issues affecting the quality of the samples. Thus, it is necessary to decontaminate the workstation as well as to use DNases- and RNases-free tools. These DNases and RNases result from paraffin block cuts used to extract the nucleic acid of interest [2].

Although some authors state that deparaffinization is not a required step [2], most protocols suggest that the material must be deparaffinized before the extraction process in order to ob‐ tain nucleic acids in a more efficient way. Most protocols use solvent (usually xylene) to re‐ move the paraffin cuts, and this procedure is followed by ethanol-based rehydration.

No matter the used protocol, digestion is the first step in the nucleic acid extraction process, and it aims to lyse membranes in order to release the cellular components. This step may be accomplished by several methods, such as elevated temperatures, enzymatic digestion, me‐ chanical disruption or even by using other detergents or according to cell type solutions. In general, enzymatic digestion with proteinase K is used in most protocols; however, the con‐ centrations and the incubation times are highly variable [9].

Nucleic acids purification is the next stage. Literature reports protocols using organic sol‐ vents (such as phenol-chloroform) [12–14], salt (salting out) [15] and other substances (Che‐ lex-100) [16] as well as protocols using commercial kits available in the market [9].

Li et al. (2008) [1] observed that RNA extraction protocols based on proteinase K digestion followed by DNase, column purification and elution treatments led to good results in FFPE samples. These authors have shown that proteinase K is essential to degrade covalently linked proteins in order to release RNA from the cell array and to inactivate RNAses, which tend to be stable.

Ribeiro-Silva and Garcia (2008) [4] have shown that proteinase K is used to degrade proteins bound to nucleic acids and that the incubation between 60°C and 70°C removes the methyl‐ ol groups added by formalin. RNA isolation by denaturing agents prevents the RNases ac‐ tion. In addition, deoxyribonuclease (DNase) incubation is required to remove the deoxyribonucleic acid (DNA) sample. Finally, purification by precipitation with alcohol po‐ rous column removes any residue and contaminants.

All tested protocols will be detailed in the sections below, as well as the changes suggested to determine the protocols that would be viable to the obtainment of nucleic acids present‐ ing adequate quality for molecular biology studies.
