Optimized High Quality DNA Extraction from Formalin-Fixed Paraffin-Embedded Human Atherosclerotic Lesions

Formalin fixation followed by paraffin embedding (FFPE) is a standard procedure for long-term preservation of pathological specimen in biobanking¹. These samples provide a valuable source for histological studies as well as molecular analyses, especially genetic studies². Further advantages of FFPE tissues are better long-term storage, lower costs, and easier storage conditions. Our intention here is to provide a reliable and easy-to-use protocol for reproducible nucleic acid isolation from small amounts of FFPE sections, since high quality DNA extraction is the first crucial step in a wide range of molecular techniques and FFPE tissues are the most available source of samples.

New scientific approaches, such as next-generation sequencing (NGS) and “omics” research approaches, require high quality of nucleic acids^3,4,5. Extracting DNA from FFPE tissue samples remains a challenging endeavor. DNA from FFPE samples can vary widely in quality and quantity depending upon its age and fixation conditions. Formalin, the most frequently used compound, leads to DNA-protein crosslinking^6,7,8 and causes unspecific random breakage in the nucleotide sequence⁹. This may significantly impact downstream genomic analyses, since crosslinking can disable polymerase chain reaction (PCR) amplification^6,10. Due to contaminants during the fixation process, purity of the DNA isolated from FFPE samples is often limited. In recent years, various methods for DNA isolation were established, mostly from cancer tissue specimens^2,11,12,13.

In general, protocols for the extraction of nucleic acids from FFPE tissue can be differentiated into three main groups. The first, most commonly used group of methods includes commercially available silica-based column systems¹⁴. The second group involves manual organic phase extraction methods with phenol and chloroform, first described by Joseph Sambrook and David W. Russell¹⁵. As a third group, automated systems were established over the last years such as liquid handling systems as well as paramagnetic particle-based systems¹⁶. Each of the three named systems holds different advantages and disadvantages such as hazardous chemicals (i.e., xylene, phenol, chloroform), high costs¹⁷, manpower¹⁸, and time consumption¹⁹. Especially, for the difficult tissue specimen as well as high throughput analyses standardization, reproducibility, relatively low time-consumption, manpower, and costs are the most relevant features in finding a suitable method for nucleic acid isolation²⁰. Automated extraction methods are known to show better reproducible results and are more sensitive for small biopsies. Moreover, less amount of tissue or blood is needed and the risk of clogging of the system due to high amounts of paraffin is reduced. Although machines for automated nucleic acid extraction and the needed kits are more expensive compared to manual methods, they are still convincing due to less problematic extraction processes. Literature search provides a lot of publications that illustrate a direct comparison between manual, column-based, and automated DNA and RNA extraction methods from different tissues and organisms, such as plants, animals, and humans as well as cells in culture^20,21,22. There are also evidences present in literature to show that DNA and RNA isolated from 10-year old snap frozen tissue can be used for downstream analyses such as PCR, quantitative PCR, NGS, methylation analyses, and cloning^{9,23,24,25,26}.

The major problem with, for example, aged human vascular tissue, as well as small tissue biopsies, especially concerning FFPE samples, is the lack of cells in the highly calcified atherosclerotic lesions, which consequently leads to low concentrations of nucleic acids¹. Although several methods for DNA extraction from FFPE tissue have already been established and are widely used, the manual sample preparation methods require long hands-on time²⁷ and toxic reagents such as xylene or phenol are necessary for deparaffinization². As described, the deparaffinization process is a crucial time-consuming step (e.g., around 30 min) that markedly affects the quality and quantity of the extracted DNA (e.g., toxic effects on DNA, such as fragmentation and degradation of deparaffinization solution and high temperatures)²⁸. Recently developed new DNA extraction protocols focus on using other non-toxic deparaffinization solutions, repair strategies and automated bead technologies. In particular, automated and semi-automated methods have been shown to be successful in DNA extraction with efficient recovery, lack of cross contamination, and easy performance²⁹. We have established a protocol that overcomes these limitations. As a result, our technique allows a reduction of processing and hands-on time at highest quantitative and qualitative standards.

Especially for reproducible high-throughput analyses such as genotyping, epigenomic studies, and RNA sequencing the handling of FFPE specimen with column-based purification systems is often difficult and time consuming (e.g., long deparaffinization steps, column clogging, and long hands-on times). Clogging of the silica membranes due to high amount of paraffin is the major issue. Other circumstances that can worsen the isolation of high-quality nucleic acids are small amounts of tissue such as micro-biopsies of skin, small mouse tissue, very fatty or calcified tissue as plaques, ossified tissue, and aged samples. Especially in diagnosis and forensics, automated and semi-automated systems such as liquid handling or paramagnetic particle-based extraction methods became more and more essential over the last few years^30,31, mainly due to relatively low hands-on times and the possibility of standardization. Most of the already published protocols work perfectly for smooth tissues with high or medium amounts of cells such as tumor biopsies or plant tissue^13,22,32. Literature about methods for semi-automated particle-based methods used for isolating DNA from relatively hard-to-handle tissue such as fixed single cells, calcified vessels, collagen rich tissue, and fatty tissue with low cell numbers are only poorly described³³.

In this study, an optimized semi-automated method for DNA isolation from vascular paraffin embedded sections is described, comparing it to two manual column-based protocols. DNA quantity, purity, and the extent of fragmentation were used for validation. The commercially available blood DNA protocol, was used as a starting point and the manual steps of the semi-automated system were subsequently optimized for the use of FFPE as well as fresh frozen tissue samples from human and animal tissue, combining steps from the FFPE and the tissue protocol. The automated step of this protocol is pre-installed on the instrument and depends on the used kit (here, the blood DNA kit). With the described semi-automated cartridge-based system it is possible to isolate DNA from blood, fresh-frozen tissue, formalin-fixed tissue and even single cells with the same protocol, machine, kit and consumables, instead of using different protocols and kits for the instrument, as it is recommended by the company. There are only minor differences in the protocols, such as one buffer and some incubation times for the different applications, which makes this protocol very useful for extracting DNA from all kinds of tissues. Our protocol is primarily optimized for calcified, poor in cells and fibrous human vascular tissue, but can of course be used and further optimized for all kinds of difficult tissues mentioned above.

Summarized, for researchers in the cardiovascular field working on atherosclerosis (e.g., aorta, carotid arteries, coronary arteries) we provide an easy-to-use, point-by-point protocol for semi-automated DNA extraction from vascular FFPE samples.