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

Summary

Here, we present a protocol for semi-automated DNA extraction from formalin-fixed paraffin-embedded lesions of human carotid arteries. The tissue lysis is performed without toxic xylene, which is followed by an automated DNA extraction protocol, including a second lysis step, binding of DNA to paramagnetic particles for cellulose based binding, washing steps, and DNA elution.

Abstract

Formalin-fixed paraffin-embedded (FFPE) tissues represent a valuable source for molecular analyses and clinical genomic studies. These tissues are often poor in cells or difficult to process. Therefore, nucleic acids need to be carefully isolated. In recent years, various methods for DNA isolation have been established for tissues from many diseases, mostly cancer. Unfortunately, genomic DNA extracted from FFPE tissues is highly degraded due to the cross-linking between nucleic acid strands and proteins, as well as random breakings in sequence. Therefore, DNA quality from these samples is markedly reduced, making it a challenge for further molecular downstream analyses. Other problems with difficult tissues are, for example, the lack of cells in calcified human atherosclerotic lesions and fatty tissue, small skin biopsies, and consequently low availability of the desired nucleic acids as it is also the case in old or fixed tissues.

In our laboratories, we have established a method for DNA extraction from formalin-fixed atherosclerotic lesions, using a semi-automated isolation system. We compared this method to other commercially available extraction protocols and focused on further downstream analyses. Purity and concentration of the DNA were measured by spectrometry and fluorometry. The degree of fragmentation and overall quality were assessed.

The highest DNA quantity and quality was obtained with the modified blood DNA protocol for the automated extraction system, instead of the commercial FFPE protocol. With this step-by-step protocol, DNA yields from FFPE samples were in average four times higher and fewer specimens failed the extraction process, which is critical when dealing with small-vessel biopsies. Amplicon sizes from 200–800 bp could be detected by PCR. This study shows that although DNA obtained from our FFPE tissue is highly fragmented, it can still be used for successful amplification and sequencing of shorter products. In conclusion, in our hands, the automated technology appears to be the best system for DNA extraction, especially for small FFPE tissue specimen.

Introduction

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.

Protocol

The permission to collect human carotid atherosclerotic specimens in our biobank was approved by the local Hospital Ethics Committee (2799/10, Ethikkommission der Fakultät für Medizin der Technischen Universität München, Munich, Germany). Written informed consent was obtained from all patients. Experiments were performed in accordance with the principles of the Declaration of Helsinki.

1. Tissue preparation

1. Prepare 5–8 tissue sections of 10 µm from the FFPE sample with the microtome and transfer it in a 1.5 mL tube. It is not necessary to reduce the excess of paraffin from the block.
   NOTE: Thinner single sections instead of one large section accelerate the buffer reaction. Discard the first sections due to O₂ exposure. For bigger samples, it is also possible to use fewer sections.
2. Centrifuge these tubes in a bench top centrifuge, set to 5,000 x g for 1 min at room temperature to collect each sample on the bottom of the tube.
   CAUTION: Too long centrifugation leads to clotting of the sample and complicates the lysis.

2. Lipid dissolution and deparaffinization

NOTE:This step is needed for deparaffinization and lipid digestion. The buffer used is less toxic than commercial deparaffinization solutions.

1. Add 300 µL of the commercially available incubation buffer and 6 µL of 1-thioglycerol to each tube.
   NOTE: Do not use more than 300 µL as this is the maximum volume of the automation system cartridge.
2. Vortex for 10 s and incubate the sample for 10 min at 80 °C and 500 rpm in a heating block to solubilize paraffin.
   CAUTION: The tissue should be completely dissolved in the end. If necessary, vortex several times during incubation.

3. Sample and protein digestion

NOTE:Native digestion with protease K is crucial to have clean DNA extracts without proteins. It also reduces any contaminating proteins present. Furthermore, nucleases are also destroyed to save the DNA³⁴. This overnight step is also needed for complete sample digestion.

1. Let the sample cool down to 60 °C and then add 30 µL of the provided Proteinase K solution.
2. Vortex again and incubate the mixture at 65 °C and 500 rpm overnight (4–20 h) in a heating block. Vortex the samples during incubation from time to time for complete sample digestion.
   NOTE: Overnight incubation leads to better results. Mixing steps are recommended every 30–60 min. In the end, there should not be any visual tissue piece inside the tube.

4. Cell lysis

1. Add 400 µL of the lysis buffer, provided in the blood kit and vortex shortly.
2. Incubate the sample again at 65 °C for 30 min with 500 rpm.
3. Let the sample cool down to room temperature. The paraffin will harden on top.
   CAUTION: Do not vortex again, to keep the paraffin separated from the sample. Otherwise, the paraffin is mixed with the tissue, which destroys the sample. The sample will be collected in step 6.1.

5. Preparation of the pre-dispensed cartridges

1. Turn on the machine, as well as the associated tablet computer.
2. Start the software app and click on the Door button to open the instrument.
3. Remove the rack from the instrument and insert the pre-filled cartridge into the probe rack. Ensure that the cartridge clicks twice when into place and remove the sealing foil.
4. Add the plunger in the last (8) well of the cartridge. It serves as pipette tip in the instrument.
5. Fill the provided 0.5 mL elution tubes with 65 µL of elution buffer, provided with the kit. Leave the tubes open and insert them into the dedicated position in the front part of the rack, after the cartridge.
   NOTE: The minimum volume for elution is 60 µL. The system will lose 5–10 µL of the added elution volume.

6. Automated DNA extraction

1. Carefully puncture the paraffin on top of the 1.5 mL tube from step 4.3 to reach to the clean sample at the bottom of the tube without mixing it with paraffin again.
2. Transfer the whole mixture (730 µL) of the prepared sample in the first well of the cartridge.
3. Insert the rack into the automated DNA extraction machine. Ensure that the rack locks in the back of the machine first and in the front afterwards.
4. Start the run by clicking the upper left orange Start button in the software. A window with different pre-installed protocols will open. Select Blood DNA protocol on the instrument. Confirm that plunger, elution tube, and sample were added by clicking yes in the software. The door of the instrument will close automatically, and the run starts (the light will turn green). The run will take approximately 38 min. No further calibration is needed.
   NOTE: Watch until the system has picked up the plungers for all samples in the rack. If this is not happening the system stops automatically and the machine protocol must be restarted.
5. Ensure that the system performs the automated lysis step in the first well of the cartridge, followed by washing steps in wells 3 to 7. There is no further programming step needed. The complete program is pre-installed by the company.
6. Once done, ensure that the system elutes the DNA in the prepared elution tubes via the added plunger. The magnetic particles stay in the plunger. The plunger in the end goes back to the last well of the cartridge.

7. Finish the run

1. When the run is completed (the machine shows green blinking light), open the instrument by clicking the button for opening (door-sign) and remove the rack from the system.
2. Discard the cartridges.
3. Reinsert the empty rack to the instrument and close the door via the door button in the upper-right corner. Close the software app and turn off the machine, as well as the tablet computer.
4. Store eluates at -20 °C for long-term storage or at 4 °C for short-term storage or use it directly for downstream analysis or concentration measurements.

Results

For the establishment of the protocol, 5 FFPE tissue blocks from patients with atherosclerosis of the carotid artery were used. DNA was isolated with an optimized semi-automated protocol (kit C) as well as with two commercially available manual column-based protocols (kit A and kit B, see Table of Materials). DNA extraction with kit A and B was performed according to the manufacturer´s protocol. The only change that was made in the protocol of the two commercially available kits (kit A and kit B): d...

Discussion

DNA extraction methods for FFPE tissue vary in quality and quantity of isolated DNA, which inevitably affects the performance of further downstream analyses. Thus, automation is becoming imperative to improve workflow and standardization, as well as quality management. Therefore, in the present study, a semi-automated method for DNA extraction from FFPE samples was evaluated demonstrating better results than the other tested manual column-based protocols.

To optimize the described semi-automat...

Materials

Name	Company	Catalog Number	Comments
1.5 ml tubes for sample incubation	Eppendorf, Hamburg, Germany	30120086
1-Thioglycerol	Promega, Walldorf, Germany	A208
Agilent tape station software 3.2	Agilent, Waldbronn, Germany
dsDNA HS Kit	ThermoFisher Scientific, Schwerte, Germany	Q32851
FFPE DNA Purification Kits (Kit A)	Norgene Biotek, Heidelberg, Germany	47400
FFPE tissue samples n=5	Munich Vascular Biobank,Munich, Germany
GeneRead DNA FFPE Kit (Kit B)	Qiagen, Hilden, Germany	180134
Heating blocks, set to 80°C and 65°C	VWR,Darmstadt,Germany	460-0250
High Sensitivity D5000 reagents	Agilent, Waldbronn, Germany	5067-5593
High Sensitivity D5000 ScreenTape	Agilent, Waldbronn, Germany	5067-5592
Incubation Buffer	Promega, Walldorf, Germany	D920
Maxwell Blood Kit RSC including: Lysis Buffer, Elution Buffer, Proteinase K	Promega, Walldorf, Germany	AS1400
Maxwell RSC 48 Instrument	Promega, Walldorf, Germany	AS8500
Microcentrifuge	Eppendorf, Hamburg, Germany
NanoDrop 2000c Spectrometer	ThermoFisher Scientific, Schwerte, Germany	ND-2000C
Optical caps	Agilent, Waldbronn, Germany	401425
Optical tube strips	Agilent, Waldbronn, Germany	401428
Pipettors and pipette tips	Eppendorf, Hamburg, Germany
Prism 6 for statistics, version 6.01	GraphPad Inc., San Diego, California
Qubit 3.0 Fluorometer	ThermoFisher Scientific, Schwerte, Germany	Q33216
TapeStation 4200	Agilent, Waldbronn, Germany
Tecan Infinite M200 Pro	Tecan, Männedorf, Swizerland	IN-MNANO