A method for improving the efficiency of DNA extraction from clotted blood samples

Background The efficient and rapid extraction of high‐quality genomic DNA from clotted blood samples, which normally have a low yield and poor quality, is an important factor in genomic research. The objective of this study was to develop a simple and safe technique for dispersing the blood clots by the ball bearing metal shots. Normally, such clot samples may not have an acceptable yield by conventional DNA extraction methods. Also, in the present study, we have further investigated to improve salting‐out DNA extraction methods. Methods Initially, 500 µL phosphate‐buffered saline (PBS) (1×) and two ball bearing metal shots were added to each tube of the clotted blood sample and then were gently rotated in an electric laboratory rotator for 1 hour at room temperature (18‐25°C). Genomic DNA was then extracted from samples using a modified salting‐out method and a modified QIAamp® DNA Blood Midi Kit and was compared with QIAamp® DNA Blood Midi Kit as a control. An assessment of the concentration and quality of the extracted DNA was performed using the UV‐visible spectrophotometer. The isolated DNA proved amenable to PCR amplification and gel electrophoresis. Results The yield and purity of DNA obtained by these three methods were significantly different (P < 0.001), with a higher yield in the modified salting‐out method. Conclusions Our proposed modified salting‐out method is simple and efficient for the isolation of DNA from old blood clot samples. It is both easy to use and is of low cost in routine laboratory tasks.

Some methods have dispersed blood clots using scalpels 17 and mesh with centrifuge 15 after separation in serum-separator tubes.
Crashing of the clot requires using sharp objects which may be hazardous for the personnel. 15 Other techniques such as using mesh with the centrifuge have limitations such as inaccurate pipetting of blood volume because of clotted blood compared to the obtained cell volume from anticoagulated blood. 15 By working on a cohort, the Mashhad Stroke and Heart Atherosclerotic Disorders (MASHAD) study, 18 where we have collected blood samples from participants dating back to more than 7 years ago, along with the fact that genomic DNA extracted from clotted blood samples normally has low yield and poor quality, and we were encouraged to improve the current extraction methods in order to be able to obtain a higher yield. In the present study, we have further developed a simple, safe, and efficient technique for the fragmentation of the clot before DNA extraction processing. We used the ball bearing metal shots as a mixture to maximize the fragmentation of the clot. Also, we have attempted to modify a salting-out method for DNA extraction with the highest possible yield from blood clots with ethylenediaminetetraacetic acid (EDTA) and tri-Sodium citrate dehydrate as a blood anticoagulant for both blood homogenization and chelated cations of Mg2 + and Ca2 + which are necessary when using DNAases. Finally, in this study, the rate of the PCR inhibitor and quality of extracted gDNA were compared between a modified salting-out method, a modified QIAamp ® DNA Blood Midi Kit, and QIAamp ® DNA Blood Midi Kit as the control group.

| Sample collection
We randomly selected thirty-one clotted blood samples collected as part of the Mashhad Stroke and Heart Atherosclerotic Disorders (MASHAD) study. The MASHAD study is a 10-year cohort study in an urban population in eastern Iran, 18 which began in the year 2010. Ten milliliters of peripheral blood was collected in plain tubes.
Samples were spun, and the serum was separated. All tubes containing clotted blood were stored at −80°C.
Another set of 32 samples of clotted blood from this cohort were chosen and considered as a comparator group. The study was extracted DNA was performed using the UV-visible spectrophotometer. The isolated DNA proved amenable to PCR amplification and gel electrophoresis.

Results:
The yield and purity of DNA obtained by these three methods were significantly different (P < 0.001), with a higher yield in the modified salting-out method.

Conclusions:
Our proposed modified salting-out method is simple and efficient for the isolation of DNA from old blood clot samples. It is both easy to use and is of low cost in routine laboratory tasks.

| Sample preparation
Before proceeding to DNA extraction using a modified salting-out method 11,19,20 and modified QIAamp ® DNA Blood Midi Kit, 500 µL phosphate-buffered saline (PBS) (1X) and two ball bearing metal shots, autoclaved in 121°C for 15 minutes, were added to each tube of clotted blood sample and were gently rotated in an electric laboratory rotator for 1 hour at room temperature (18-25°C). In QIAamp ® DNA Blood Midi Kit, blood samples were thawed for 1 hour at room temperature (18-25°C).

| Protocol A: modified salting-out method
Two milliliters of blood clot was transferred to a tube containing 7 mL of cell lysis buffer solution (CLB) (0320 mmol/L sucrose, 10 mmol/L Tris-HCl, 2 mmol/L MgCl 2 , 1% Triton X-100, 4 mmol/L tri-Sodium citrate dehydrate) (pH 6.50) (Merck, Germany), and the tubes were mixed well by pulse-vortexing for 2 minutes. After centrifuging at 3800 rpm (1533g) for 10 minutes, the supernatant liquid was carefully discarded to waste and 5 mL CLB was added to pellet again, and tubes were well shaken for 2 minutes and centrifuged at 3800 rpm

| DNA yield and quality
Two microliters of purified DNA solution was used to measure the quality and quantity of the DNA (ng/μL) (A260/A280) by UV-visible spectrophotometer (BioTek, USA).

| Agarose gel electrophoresis
In order to assess DNA degradation and the molecular weight of the DNA by each method, gel electrophoresis was performed by loading 3 μL of extracted DNA on 0.2% agarose gels (Max Pure, Iran) prepared in 0.5X TBE buffer (Pars tous, Iran).

| RE SULTS
The mean amount of DNA extracted by each method is shown in Table 1. The mean yield and purity of DNA obtained by these three methods were significantly different (P < 0.001). A significant difference was also observed in the A260/A280 ratios among those methods (P = 0.01). However, when performing the statistical analysis (Mann-Whitney U test for pairwise comparisons), statistically significant differences were not found for either of the gDNA yield (P = 0.4) TA B L E 1 Yield and A260/A280 ratio of DNA purified from clotted blood   Table 2.

| D ISCUSS I ON
In previous studies, the physical breakage of dispersing blood clots was carried out by different methods. The sterile scalpel and mesh were used for a quick isolation of DNA from clotted blood samples. 15,16,21,22 This study developed an alternative method to process a large number of blood clots for isolation of high-quality DNA, which involves the use of the ball bearing metal shots to homogenize and break down the blood clot, followed by adding phosphate-buffered saline (PBS) to maximize the volume of blood in liquid form. It is believed that PBS will increase the buffy coat volume. 23 Our results showed a significant increase of DNA extraction and (e) improving the safety as there are no sharp objects used and human intervention as well as exposure to hazardous materials is minimized.
Regarding evaluation of the extracted DNA, Figure 2 indicates that the extracted DNA was successfully amplified via PCR genotyping. The quantification of gDNA was more accurately determined using real-time PCR. We did not identify prominent inhibitory factors in the solution. These findings demonstrated the quality and quantity of DNA samples purified from clotted blood, and this method would also help to use frozen blood clots as a source of DNA in many areas of molecular biology.
In practice, the use of the ball bearing metal shots to homogenize and disperse the blood clot followed by a routine method for extraction of DNA is simple and safe. The advantage of the modified salting-out method is simple and inexpensive ( Table 2) Table 3. It shows the reduction of time required for DNA extraction, and improvement of the quality and quantity of DNA is fairly reduced.
In conclusion, the results showed that this method is simple, safe, and more economical for isolation of DNA from frozen blood clots.
Although this method is beneficial and practical for either fresh or clotted blood, the ball bearing metal shots for the fresh blood, which is a homogeneous suspension, may be skipped.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.