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

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): deparaffinization in the beginning was performed twice due to high amounts of paraffin surrounding the sections. After deparaffinization with xylene, DNA was bound to the column-membrane and washed three times. After drying the membrane by centrifugation, DNA was eluted with 60 µL of the provided elution buffer.

To determine the optimal quantity of samples needed for reproducible results from every patient, different amounts of material were taken. Consecutive tissue sections of 2 x 10 µm (20 µm in total), 5 x 10 µm (50 µm in total), and 8 x 10 µm (80 µm in total) were used (Figure 1). The quality and quantity of total genomic DNA isolated with the automated system and the two-manual column-based systems were assessed both with spectrometry, a microplate reader spectrometric system as well as fluorometry. Fragmentation analysis was performed using automated screen-tape technology and polymerase chain reaction (PCR). Statistical analysis was done using commercially available software (see Table of Materials).

The semi-automated protocol leads to higher quantity of the extracted DNA
Using micro-volume spectrometry and plate reader spectrometry as well as fluorometry as shown in Figure 2, the total amount of genomic DNA was compared for the different amounts of tissue sections (20 µm, 50 µm, 80 µm in total). A linear increase of DNA concentration was obtained with the cartridge-based semi-automated kit C, with all three quantification methods. For the manual kits A and B, no significant differences were observed between 20 µm, 50 µm, and 80 µm sections. The mean concentration of all five tissue samples measured with micro-volume spectrometry and microplate reader technology for DNA extracted with the semi-automated protocol (Figure 2A,B, Table 1) showed a significant, four times higher, concentration (45 ng/µL) compared to the column-based protocols A and B (p = 0.016). Kit A attained mean concentrations of 12.5 ng/µL, while Kit B attained only 8.5 ng/µL. Figure 3 shows the quality of the extracted DNA with the three named methods. Summing up that there is no big difference in quality. The semi-automated kit shows more stable quality, the measured quantities are comparable. Similar results were found by using fluorimetric measurements of the DNA concentration (Figure 2C). Mean concentrations of DNA using kit A were further reduced to 3 ng/µL compared to the spectrometric analysis. Summarizing the quantity of DNA, the automated protocol turned out to be the most efficient method for low cells-tissue samples.

Automated extraction protocol leads to a better quality of the extracted DNA
Furthermore, the quality of DNA isolated with the three different protocols was assessed spectrophotometrically, both using spectrophotometer and microplate reader, calculating the A260/280 ratio (Figure 3A,B and Table 2). Nucleic acids have their absorbance maximum at 260 nm. Purity of high quality DNA is generally in the range from A260/280 values of 1.6–1.8, as evidenced by the two dashed lines in Figure 3A,B³⁵. The mean ratio of the manual column-based kit A was 1.92 ± 0.6 in spectrometry and 2.58 ± 1.2 measured with microplate-reader technology. Kit B showed smaller ratios (1.45 ± 0.3 and 1.85 ± 0.7). DNA isolated with the semi-automated kit C showed purity ratios of 1.4 ± 0.07 in spectrometry and 1.68 ± 0.05 calculated with the microplate reader. In both the measurements, the automated isolation method showed less variation in purity ratio than the manual kits but only kit A showed significant differences in paired t-test analysis because of the huge scattering (p = 0.0025 and p < 0.001). Regarding the microplate measurement, all samples isolated with the automate system passed the purity criteria (ratios between 1.6 and 1.8), whereas kit A and kit B in many cases failed these criteria (p < 0.001). Spectrometry (Figure 3A) evidenced that not all samples isolated with the optimized cartridge system (minimum ratio = 1.36) reached the defined purity threshold (dashed lines, indicating the A260/280 ratio of 1.6 and 1.8), indicating buffer contamination. In addition, huge variation of the calculated ratio for kits A and B was observed. Consequently, semi-automated DNA isolation provides samples with comparable and reproducible quality, whereas the DNA quality is widely fluctuating with column-based isolation methods. Another quality check was performed using DV200 calculation (Figure 3A, Table 2). Here, the manual column-based extraction system A showed a value of 90% ± 3.8 for fragments bigger than 200 bp, whereas kit B only showed 85% ± 7.1 (p = 0.01). Also, in this analysis, DNA extracted with the semi-automated protocol showed the highest percentage of fragments bigger than 200 bp (92% ± 3.6).

Summing it up, quality of the DNA extracted with the semi-automated system was more stable and reproducible, whereas DNA isolated with the column-based systems showed more variation in quality ratios. DV200 indicated similar quality for kits A and C, whereas kit B had less fragments bigger than 200 bp.

DNA fragmentation is independent of the used protocol
Fragmentation of the DNA was assessed with semi-automated screen tape technology. In Figure 4A, representative gels are shown to display the extent of fragmentation of all samples used in this study. Dark color indicates a higher density whereas grey shaded images indicate lower densities. Here, similar patterns were observed for all samples and in all three kits. The first three samples show the darkest region (which equals the highest accumulation of the fragmented DNA), in the lower part of the gel representing the smaller DNA fragments, samples from patient 4 and 5 show more (denser) fragments in the upper part of the gel, indicating less fragmented DNA. In Figure 4B, the difference in DNA fragmentation using various amounts of material (20 µm, 50 µm, and 80 µm) is shown for one representative sample using all three isolation strategies. Electropherograms show the corresponding fragment distribution. The data is summarized in Table 3. Here, the only visual difference between the three kits was found in the graphs of the 20 µm sections, with the automate kit showing a clearer electropherogram with more DNA detected. For 50 µm and 80 µm sections, no visible difference in the electropherogram was found between the different kits used in the study.

Figure 5A shows the mean distribution of the fragment sizes for the semi-automated cartridge system kit C as well as for the two manual kits A and B. Mean distribution of the fragment sizes for the automated system shows the following results: 5% of the DNA fragments were smaller than 200 bp, 62% sized between 200 bp and 1500 bp and 29% of the DNA fragments were longer than 1,500 bp. Higher percentage of augmented DNA fragmentation was measured with Kit B (12%) and Kit A (8%). Medium size fragments were similar for kit A (62%) and kit B (57%). Regarding the longer DNA fragments, no significant difference (kit A 26%, kit B 27%) were observed. This leads to the conclusion that DNA isolated with the optimized semi-automated kit (kit C) seems to be less fragmented than DNA isolated with column-based kits.

The fragmentation peak (highest amount of DNA for a certain fragment size) in all three kits was analyzed for each of the five tissue samples (Figure 5B). No differences between the three isolation methods could be detected concerning the overall DNA fragmentation. Of note, regardless of the kit utilized, samples 1 to 3 show an average maximum peak between 280 bp and 620 bp whereas samples 4 and 5 are less fragmented with an average fragmentation peak between 1,200 bp and 1,800 bp. These results indicate that DNA fragmentation is affected by the quality of the specimens itself.

DNA extracted with the semi-automated kit is suitable for PCR analysis
Ultimately, eight DNA samples isolated with the optimized protocol were used for downstream analysis performing PCR of different fragment lengths. Appropriate primers for the beta-actin housekeeping gene were chosen. A primary smooth muscle cell line served as a control (Figure 6). By displaying the detectable fragments in agarose gel electrophoresis, both samples with large maximum fragments (500 bp–800 bp for samples 1, 2, and 8) as well as samples with only 200 bp of maximum fragments were detected (samples 6 and 7), independent of the extraction method used.

Figure 1: Experimental workflow. The main steps of sample preparation and semi-automated DNA extraction are shown in the context of the used specimens and in comparison, with the manual DNA extraction kits A and B. Please click here to view a larger version of this figure.

Figure 2: Concentration measurement. The upper part (A-C) shows the DNA concentrations measured with spectrometry, microplate reader technology and fluorometry using 20 µm, 50 µm, and 80 µm sections (each, n = 5) for all three different kits applied. The lower part shows the distribution of the DNA concentrations of five samples (80 µm) measured with spectrometry (D), microplate reader technology (E) and fluorometry (F). p-values are calculated with the paired t-test, p < 0.05 was considered significant. Please click here to view a larger version of this figure.

Figure 3: Purity of the isolated DNA. The purity of DNA was assessed by calculating the A260/280 ratio in two different spectrometers (A,B) for all three different kits (n = 15). Dashed lines demonstrate the optimal ratio (1.6–1.8). (C) shows the DV200 (fragments >200 bp) in % calculated via fragmentation analysis. P-values are calculated with Mann-Whitney test and shown in the graph accordingly, p < 0.05 was considered significant. Please click here to view a larger version of this figure.

Figure 4: Representative images from the fragmentation analysis. The fragmentation of each individual sample 1–15 is shown in the screen-tape traces. For every sample (n = 5) different amount of DNA from 20 µm, 50 µm, and 80 µm sections were analyzed separately (n = 3 for each sample) (A). (B) shows the DNA fragmentation of one representative specimen for 20 µm, 50 µm, and 80 µm sections for all three kits. Please click here to view a larger version of this figure.

Figure 5: Fragment distribution by screen-tape analysis. (A) shows the distribution of short, medium, and long fragments within the samples isolated with different kits. (B) shows the maximum fragmentation peak of the DNA isolated from samples 1–5 plotted for the individual kits. For every kit, the 20 µm, 50 µm and 80 µm sections of each samples are pooled. p-values are calculated with paired multiple t-test and shown in the graph, p < 0.05 was considered significant. Please click here to view a larger version of this figure.

Figure 6: Fragmentation analysis with PCR. This gel electrophoresis shows the PCR products with different amplicon lengths of the beta-actin housekeeping gene for eight different samples and one control cell line. Lanes are named as follows: 1 = 100 bp, 2 = 200 bp, 3 = 300 bp, 4 = 400 bp, 5 = 500 bp, 6 = 600 bp, 7 = 700 bp, 8 = 800 bp, 9 = 1,000 bp, and 10 = 1,300 bp. Please click here to view a larger version of this figure.

Table 1: Results of quantitative analysis. This table shows the minimum, maximum, and mean concentration, of the DNA isolated from the five patients with the three different kits. Concentrations were measured spectrometrically using micro spectrometer and microplate reader, as well as fluorometrically.

Table 2: Results of qualitative analysis. A260/280 ratios calculated with micro-spectrometer as well as microplate-reader are shown for every sample (n = 15) and the three different extraction methods. DV200 (fragments bigger than 200 bp) calculated via fragmentation analysis is also shown for kits A, B, and C.

Table 3: Results of fragmentation analysis. Here, the percentage of the fragments smaller than 200 bp, bigger than 1,500 bp and between 200 bp and 1,500 bp is shown for all five patients, the different amounts of starting material (20 µm, 50 µm, and 80 µm) and the three different isolation methods. Percentages smaller than 2% were named as not detectable.