Journal of Pharmacy and Pharmacology 5 (2017) 554-560

doi: 10.17265/2328-2150/2017.08.010
D DAVID PUBLISHING

Isolation of Cell-Free DNA from Seminal Fluid

Tina Draškovič
Faculty of Pharmaciy, University of Ljubljana, Ljubljana 1000, Slovenia

Abstract: Cell-free DNA (cfDNA) is small short double stranded molecule that is also found in seminal fluid. It is a product of
apoptotic cells in different developmental stages during spermatogenesis. To final concentration of total cfDNA in semen contributes
also cfDNA secreted from living cells and cfDNA that is result of different diseases e.g. prostate cancer or infertility. Amended
concentration (high or low) can be connected to prostate cancer or male infertility and can represent important non-invasive diagnostic
biomarker for detection and prognosis of these pathological conditions. In this paper, I will discuss different approaches for isolation of
cfDNA from seminal fluid, which includes selection of the samples, separation, isolation, extraction, purification and analysis. Today’s
most popular approach for isolation is the use of commercial kits based on selective binding and elution on silica-membrane technology,
magnetic-bead technology or extraction with organic solvents and salting out procedure. Furthermore I will present that I tried to isolate
cfDNA from semen with QIAamp DNA Mini Kit to confirm the presence of cell-free DNA in our samples. In the end I will describe
problems we are facing during cfDNA measurement which are mainly associated with low concentration of cfDNA in samples.

Key words: Cell-free DNA, seminal fluid, diagnostic biomarker, prostate cancer, male infertility, cell-free DNA isolation, problems of
cell-free DNA measurement.

1. cfDNA non-invasive diagnostic biomarker for detection and

prognosis of prostate cancer and male infertility (sperm
1.1 Biological Aspects of cfDNA
quality) [1]. Other applications can longitudinally
The cfDNA are mostly double-stranded molecules monitor disease progression and response to treatment
with molecular weight significantly lower than [3]. In different studies cell-free DNA was detected in
genomic DNA. Range of wide is from 0.18 kB to 21 kB all semen specimens [2]. Isolated cfDNA from semen
[1]. In different studies cell-free DNA was detected in represents easily accessible and non-invasive source of
all semen specimens [2]. Scientist predicts that this is a genetic material for further analysis [3].
result of prominent apoptosis during spermatogenesis
1.3 Prostate Cancer and cfDNA
(e.g. spermatocytes and spermatids [2]) which results
in high concentrations of cfDNA in sperm [3] with Factors that can influence cfDNA changes in cancer
heterogenic size molecules [2]. Cell-free nucleic acids patients are tumor stage, tumor grade, tumor size,
(cfDNA and RNA) are released by dying cells, actively tumor aggressiveness and metastasis which can lead to
secreted by living cells and may have biological role in quantitative and qualitative changes [4].
the organism [3]. Generally higher cfDNA concentrations are found in
Data: concentration of cfDNA in seminal plasma of cancer patients because of its release from tumor cells
normozoospermia: 1.34 ± 0.65 μg mL−1, with ranges and also non-tumor cells. Due to previous researches,
from 0.51 to 2.73 μg mL−1 [3]. cfDNA in prostate cancer patients could be associated
with tumor stage, Gleason grade, metastatic spread and
1.2 Clinical Application
PSA (prostate-specific antigen). Higher concentrations
Cell-free DNA can be used as important of cfDNA can be informative and used as independent
factors for poorer outcome regarding survival or
Corresponding author: Tina Draškovič, dipl. ing. lab. disease-free interval [4].
biomed., research fields: medicine, biochemistry.
 Isolation of Cell-Free DNA from Seminal Fluid 555

Improvement of predictive value for prostate cancer We should separate each sample in more aliquots—one
can be achieved with adding cfDNA in multivariate or more for analysis and one which should be frozen up
model with age, PSA, %PSA and prostate volume [4]. to at least 6 months for later investigation or repetitions
Regarding this cfDNA can be suggested as if needed [2].
independent predictor for development of prostate
3. Separation
cancer.
Different changes may occur in cfDNA in cancer Cells from seminal plasma must be separated from
patients including changes in fragmentation (size), sperm supernatant—we can achieve that with
mutations in tumor suppressor genes and oncogenes, centrifugation: low speed centrifugation to avoid cell
microsatellite alterations and hypermethylation of lyses (e.g. first 400 x g for 10 min and then seminal
different genes. In different studies scientist came to plasma again on 200 x g for 20 min) followed by
different conclusions. In one study scientist did not filtration and ultracentrifugation or high speed
found correlation between fragmentations of cfDNA. centrifugation (16,000 x g for 5 min) [1]. Main
Conversely the other study shows that there are higher advantage of high speed centrifugation is that seminal
concentrations of small cfDNA fragments in prostate plasma can be used for directly cfDNA while sample
cancer patients. These short fragments can be useful for after centrifugation at low speed need to be further
diagnosis and prognosis of prostate cancer [4] and are treated [1].
suggested as a cancer marker [2].
3.1 cfDNA Isolation/DNA Extraction and Purification
1.4 Men Fertility and Cell-Free DNA
Nowadays, there is a wide selection of known
One study shows that only low-molecular weight techniques with many modifications for isolation of
cfDNA in seminal plasma is related to specific sperm cfDNA. Most widely used are numerous kits for
parameters important for men fertility. Due to that extraction of cfDNA from blood (plasma and serum),
Low-weight cfDNA could represents marker of semen urine and other body fluids among which we can also
quality and fertility-related problems in the male count seminal fluid. The most used are different
reproductive system [2]. We could assume that low commercial kits from different producers. Some
concentrations are consequence of reduce amount of manufacturers are QUIAGEN, Macherey-Nagel
cells in all or specific developmental stage during (NucleoSpin kits), EpiGentek, Thermo Fisher
spermatogenesis which results in lower concentration Scientific and many others. These kits are based on
of released cfDNA. selective binding and elution on silica-membrane
technology, magnetic-bead technology or extraction
2. Samples
with organic solvents and salting out procedure. After
For standardization of method Semen donors must isolation cfDNA is suitable for a broad range of
be men with normozoospermia (following WHO downstream applications, including next-generation
guidelines). Sexual abstinence of donors is required [1]. sequencing, real-time PCR, digital PCR and others.
Also routine andrological analyses of semen must be Great importance is that we use isolation methods
performed—cells should be negligible and round. that capture all of DNA fractions [4].
Predictive parameters of men fertility should be
3.2 Silicone Column Binding
examined: concentration, rapid progression, strict
normal morphology, heat-included hyper activation These commercial kits are based on silica-gel
and capacity index from the sperm penetration assay. membrane technology by selective binding and
556 Isolation of Cell-Free DNA from Seminal Fluid

stepwise elution of DNA [4]. During the procedure to capture fragments of all sizes. Moreover when
DNA is reversible bind onto silica membranes, extraction is performed automatically the scientist does
particles or beads at defined pH and high concentration not have insight in procedure. Manipulation of
of chaotrophic salts. The binding is result of interaction different conditions is therefore not possible.
between positive silica particles and negative charged
3.3 Extraction with Organic Solvents E.G.
DNA. In next step, contaminants are washed away and
Phenol-Chloroform Separation and Salting-Out
with the application of low salt buffer we elute and
Procedure
collect the DNA [5].
We can use Automated/manual kits e.g. QIAamp There are many known protocols of
DNA blood mini kit or QIAamp circulating nucleic phenol-chloroform extraction e.g. protocols according
acid kit. The latter is from QUIAGEN with following to Schmidt et al. [8], Yuan et al. [9], and Hufnagl et al.
capillary gel electrophoresis. [10], and a THP (triton/heat/phenol) protocol
This approach is popular for isolation of high-quality according to Xue et al. [11].
DNA via adsorption chromatography [5]. In one study I will describe protocol from Thermo Fisher for
Modified Isocratic Capillary electrophoresis was used. Phenol/Chloroform Extraction followed by Ethanol
Agarose gel inside the capillary tube was to enhance Precipitation. First we have to add one volume phenol:
detection of trace amounts of cfDNA [2]. chloroform: isoamyl alcohol to my sample and mixed
Biggest pro for this method is time—usage of by vortex or with hand. Next step is centrifugation
commercial kits and automated techniques represents after which we carefully remove aqueous layer and
fastest way of cfDNA isolation. They are also easily transport it into new tube. Lastly ethanol precipitation
available and can be ordered on internet. is performed. We add Glycogen, NH4OAc and ethanol
Manufacture’s protocols guide the user throughout the to the aqueous phase and leave mixture on -20 °C
whole process. Therefore the use is simple. We can overnight to achieve DNA precipitation. We can store
isolate DNA from very small volume of sample and sample at -80 °C or centrifuge them to get the pellets of
also obtain concentrated DNA from diluted samples [6]. DNA. We carefully remove the supernatant and add
Manually we can do this with binding-washing-elution ethanol. We use centrifugation again and remove
procedure on DNA purification column. This kind of supernatant. In the end we have to dry and resuspend
DNA purification column is easy to manipulate the DNA by pipetting and centrifuging briefly to
therefore can be suitable for different applications in collect the sample and place it on ice [12].
laboratories. Isolation time of cfDNA is approximately According to the different studies, previously
30 minutes [1]. mentioned procedures have higher recovery of DNA
The main problem when using commercial kits it`s than ones using binding columns [7] e.g. salting-out
that cfDNA yield can vary to 50%. This is because of DNA isolation procedure has higher recovery as
the loss of short DNA fragments (< 100 bp) during the commercial kits because of loss of small fragments of
isolation process. Fragments do not bind to column or cfDNA during the isolation process [1]. It was
bind to tight. Consequently they cannot be eluted from confirmed that greater amount of small fragments of
the column [7]. This represents great problem, because cfDNA was isolated with this method. Another
this fragments are usually most informative and advantage is that a lot of steps in the procedure can be
contain different mutations especially in cancer adjusted. Therefore, we can gain greater recovery and
patients [1]. In the future scientists should try to higher efficiency with manipulation of specific
develop columns and commercial kits that will be able conditions.
 Isolation of Cell-Free DNA from Seminal Fluid 557

One of main drawbacks is that methods are proceed with cfDNA. We followed the Protocol for
time-consumable and are not so easy to perform in isolation of genomic DNA from sperm using the
comparison with other methods e.g. binding kits. QIAamp DNA Mini Kit.
Before starting we had to equilibrate sample and
3.4 Magnetic Beads
buffer AE in room T and prepare Buffers AW1, AW2
Principle of this method is that target DNA binds on and Buffer X2 by following recepies:
magnetic beads/particles, coated with Antibodies AW1: 19 mL AW1 concentrate + 25 mL Ethanol
which bind DNA or have surface that reversibly (96-100%)
interacts only with DNA (e.g. silica). After incubation AW2: 13 mL AW2 concentrate + 30 mL Ethanol
of sample with magnetic beads, we separate the beads (96-100%)
with bound DNA in magnetic field. Other Buffer X2: 20 mM Tris Cl (pH 8), 20 mM EDTA,
contaminants stay in solution and can be selectively 200 mM NaCl, 80 mM DTT, 4% SDS, 250 µg/mL
removed. In the end, the beads are washed and DNA is Proteinase K (just before use)
eluted and ready for further use [13]. Then we perform isolation following these steps:
On the market we can found kits from different (1) Into 1.5 mL tube we add 100 µL sperm plasma
manufactures with this technology e.g. The EpiQuik™ and 100 µL buffer X2, mixed and incubate at 55 °C for
Circulating Cell-Free DNA Isolation Kit (1), or at least 1 h with occasionally mixing.
MagMAX Cell-free DNA Isolation kit. (2) Add 200 µL Buffeer AL and 200 µL of ethanol
The major advantage is that there is no need for and mix by vortexing
sample preparations with centrifugation or vacuum Then we follow regular procedure of step 5 to 8 of
[13]. Moreover there is absence of the precipitation the tissue Protocol of the QIAamp DNA Mini Kit
step (which often compromises yield and purity). (3) We apply the mixture including precipitating
Sample input volume can vary the high sample into spin column (in a 2 mL collection tube)—we had
volumes and minimize sampling error [14]. Samples to pay attention that we do not wett the rim. After
can be processed automatically, semi-automatically or closing we centrifuged the column with sample at
manually and with automated techniques we can 6,000 x g for 1 min. We placed QIAamp column in 2
achieve high-throughput applications [5]. Technology mL collection tube and discard the tube containing
enables reproducible recovery of high-quality DNA. filtrate.
Isolated DNA can be used and analyzed with different (4) After opening of the spin column we add 500 µL
quantification and qualification methods. This Buffer AW1, closed and centrifuge at 6,000 x g (8,000
approach is relatively fast and DNA can be directly rpm) for 1 min. We placed column in 2 mL collection
used [15]. This method is known by its simplicity [5]. tube and discarded tube with filtrate.
A con of this method is variability in the efficacy of (5) We opened and added 500 µL Buffer AW2,
extraction. It also required specialized instruments closed and centrifuge by full speed (20,000 x g; 14,000
which can represent additional cost [5]. rpm) for 3 min. In this step we could use new collection
2 mL tube and centrifuge at 20,000 x g gor 1 min.
4. Practical Work
(6) We placed spin column in 1.5 mL micro
In our research, we used QIAamp DNA Mini Kit for centrifuge tube and discarded tube with filtrate. After
isolation of cell-free DNA from sperm. Firstly, we tried adding of 200 µL Buffer AE or distilled water we
to isolate whole DNA in sperm to confirm the incubated mixture for 1 min at room Temperature and
effectiveness of used method. In the next step we centrifugated at 6,000 x g (8,000 rpm) for 1 min.
558 Isolation of Cell-Free DNA from Seminal Fluid

(7) Lastly we eluted the DNA in 50-100 µ Buffer AE For measurement of extraction efficiency, fragment
or distilled water. size bias and yield for validation of cfDNA methods,
we need quality controls. In one study they used means
5. Analysis
of improved reporting of cfDNA yield by comparing
For quantification in different studies we can use quantitative measurements of different reference gene
different approaches. I will mention only some of assays in plasma samples and validating this with
them: digital PCR [4].
(1) Real-time qPCR—e.g. real-time PCR targeting
6. Preanalytical and Analytical Problems of
hGDF gene [2] or human genomic targets telomerase
cfDNA Measurements
reverse transcriptase (TERT) or other genes. It would
be advisable to measure concentration of multiple While working with cell-free DNA, we face with
reference genes for more reliable results of total different problems. One of the main reasons for that is
cfDNA quantity [4]. For visualizing we can use low concentration of cfDNA in samples. Rapid
different dyes e.g. SYBER GREEN I and use its decrease in its concentration after release and sampling
fluorescence for detection of amplified products, is also a problem. In the following, I will highlight
acquiring melting curve for uniformity and calibration some preanalytical and analytical problems scientists
curve. One possibility for getting calibration curve is are facing when working with cfDNA:
the use of concentrated cfDNA in serial dilutions. With  Sampling and processing (time interval between
this technique, we can also estimate the size collection and centrifugation, storage (temperature,
distribution/fragmentation of cfDNA [2]. time), centrifugation time and forces…);
(2) Digital PCR  Different alternative protocols for isolating
(3) Droplet digital PCR cfDNA—high variability between studies;
It is recommended that for all of PCR assays targeted  Different measurement principles—simple
sequences are as short as possible [1]. We can compare spectrophotometric method or sensitive fluorometric
quantitative PCR measurements of different reference approach using different dyes (PicoGreen, SYBER
gene assays and validation with digital PCR. Green I), different PCR assays with different targets.
(4) NGS (Next-generation sequencing) [3] (e.g. telomerase, beta-actin, beta-globin) [1];
One important aspect of isolated cfDNA is size  Lack of standardization, appropriate controls, and
distribution/fragmentation of it. It can be accessed with reference materials [4];
electrophoresis. In that manner, agarose gel can be used
7. Conclusions
for determination of size distribution after use of gel
dyes e.g. staining-SYBR-Gold fluorescent stain and The potential of cfDNA in seminal fluid as a prostate
following ultraviolet transillumination for visualization cancer biomarker as well as a biomarker for diagnose,
[1]. Fluorometric assay is recommended because is prognosis and monitoring in conditions related to mane
simple, robust and enough sensitive. Detection limit for infertility is becoming increasingly apparent. Different
this technique is around 1 ng/mL. We can also use studies show that cfDNA present in semen and its
DNA marker to track cfDNA fragment sizes [2]. fragmentation is correlated to important sperm
We can also investigate the linearity of extraction parameters linked to normal sperm function and
efficiency—with applying different input volumes of presence of prostate cancer.
seminal fluid (e.g. 1, 2, 3 and 5 mL; 3 independent I tried to develop a method for extraction of cfDNA
sets). from semen. In article I described use of QIAamp
 Isolation of Cell-Free DNA from Seminal Fluid 559

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