Journal of Animal Science Advances
Effect of Storage Conditions on DNA Quality of Primary Sex
Organs
Ranjhani. A., Khatri. P., Bhutto. B., Memon. A. A., Shahnaz. I., Sheeraz. M. and Rao I.
J Anim Sci Adv 2014, 4(10): 1059-1067
DOI: 10.5455/jasa.20140613111535
Online version is available on: www.grjournals.com
ISSN: 2251-7219
RANJHANI ET AL.
Original Article
Effect of Storage Conditions on DNA Quality of Primary
Sex Organs
Ranjhani. A.,  Khatri. P., Bhutto. B., Memon. A. A., Shahnaz. I., Sheeraz. M. and Rao I.
Sindh Agriculture University Tandojam, India.
Abstract
In order to optimize a rapid and cost-efficient genomic DNA extraction protocol and to evaluate the effect
of extracted DNA stored on various temperatures, 100 fresh tissues of primary sex organs of Kundhi buffalo
were collected from local slaughter house. The DNA was extracted using commercial kit, its modified methods
and phenol-chloroform method (protocol-4), the fresh and stored DNA was measured through
spectrophotometer (Nanodrop 1000). The DNA stored at -20°C, +4°C and at room temperature was degraded in
all types of methods of DNA extraction in both types of tissue, the significantly higher degradation was found
in the samples when stored at room temperature. In conclusion the both phenol-chloroform and commercial kit
yielded a good quality DNA. Genomic DNA stored at -20°C was of good quality, whereas, the storage at room
temperature showed degradation more rapidly than other storage conditions.
Keywords: DNA, ovary, testes, buffalo, temperature
Corresponding author: Sindh Agriculture University Tandojam, India.
Received on: 28 Mar 2014
Revised on: 05 Jun 2014
Accepted on: 13 Jun 2014
Online Published on: 30 Oct 2014
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J. Anim. Sci. Adv., 2014, 4(10): 1059-1067
EFFECT OF STORAGE CONDITIONS ON DNA QUALITY OF PRIMARY SEX ORGANS …
Introduction
Stabilization of intact biological evidence in
cells and the DNA extracts from them is important;
DNA samples may be needed in casework for
replicate testing, confirmation of results, and to
accommodate future testing with new technologies
(Lee et al., 2012).
Storage of DNA evidences either in the original
sample or derived in the form of an extract is an
important issue in forensic and other molecular
biological experiment requires DNA analysis.
Frozen storage methods are effective at maintaining
DNA samples over long periods of time; they are
limited by increased cost and potential sample loss
due to power failure or unsuccessful transport (Uren
and Patel 2012). DNA sample storage has very
much importance particularly in diagnosis and
research in various veterinary clinics and
laboratories. In case of reconfirm the challenging
results and criminal cases the stored DNA may be
used for further investigation and analysis. Dry
storage of nucleic acids has been recommended to
eliminate the need for cold storage based on the
assumption that nucleic acids are stable when dry.
However there are numerous examples where
degradation occurs during storage, in the cold or at
ambient conditions that can irreversibly damage
samples in solution (Anchordoquy et al., 2007).
Although DNA can be dried without serious
damage in the short term, it is nevertheless
imperative to prevent chemical degradation and
aggregation for optimal recovery of samples.
There is controversy regarding DNA damage
resulting from repeated cycles of freeze-thawing
and it is common practice to store DNA in aliquots
to minimize the number of times the DNA is
thawed. The modification of DNA method
improves the yield and purity of DNA is useful to
laboratories performing DNA based diagnostic
work or studying molecular genetic mechanisms of
disease (Ahmad et al., 1995). Environmentally
challenged, degraded or damaged samples
(Budowle et al., 2009; Budowle and Daal 2009) of
DNA may further not be used for down streaming
applications like PCR. Storage of DNA at –20°C to
–80°C may well provide adequate conditions
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depending on the quality and quantity of DNA
desired and the time frame in which the sample will
be stored. To avoid chemical and enzymatic
degradation, DNA is often stored as a precipitate in
ethanol at –80°C. Under these conditions, nucleic
acids are stable for prolonged periods (Qiagen
2009). However, neither of these conditions will
maintain DNA quality equivalent to maintenance at
liquid nitrogen temperatures over extended time
periods, however, in a laboratory setting the DNA is
most commonly stored at 4°C, –20°C or –80°C.
The ability to stabilize and protect DNA of
various tissues from degradation at ambient
temperatures for extended time periods could have
tremendous impact in simplifying and improving
sample storage conditions and requirements. This
study is designed to evaluate the stability of DNA
of buffalo primary sex organs extracted by various
methods stored at various temperatures.
Materials and Methods
Sample Collection
One hundred samples from each ovary and
testes of Kundhi buffalo were collected from local
slaughter house of Tandojam and Hyderabad. After
collection samples were immediately wrapped into
aluminum foil than kept in ice and transferred to the
laboratory of Department of Animal Reproduction,
Faculty of Animal Husbandry and Veterinary
Science, Sindh Agriculture University Tandojam.
10 primary sex organs from each female and male
animal were studied, the samples included in study
were properly examined and confirmed for normal
organs i.e. free from any pathological changes like
cystic ovaries, orechitis etc. The selected organs
were transferred to the laboratory of Veterinary
Parasitology Department, Faculty of Animal
Husbandry and Veterinary Science, Sindh
Agriculture University Tandojam for further
experiments.
DNA Extraction through Commercial Kit
Before extraction of DNA, all materials like
collection tubes, pipettes and glassware were
sterilized properly to ensure the prevention of
contamination. For DNA extraction about 20 mg
tissues from testis and ovary of adult buffalo was
J. Anim. Sci. Adv., 2014, 4(10): 1059-1067
RANJHANI ET AL.
taken with the help of sterilized scalpel and
transferred into eppendorf tube. The obtained
sample was further broken in to small parts and
minced with the help of sterilized blade. After
breakdown of samples, the DNA was extracted
through commercial kit (Gene JET Genomic DNA
Purification Kit #K0721, (Fermentas, Thermo
Scientific),
according
through
manufacture
instruction (Protocol 1). Briefly, the 20mg samples
were homogenized and transferred into individual
1.5ml eppendorf tube and 180µl digestion buffer
was added in each of the tube and were resuspended. After re-suspension 20µl Protienase K
was added and mixed thoroughly by vortexing till a
uniform solution was obtained. The samples were
stored at 56°C for 24 hours in incubator until
complete lysis occurs. After that 20µl of RNase A
solution was added and were vortexed, the samples
were incubated for 10 minutes at room temperature.
200µl of lysis solution was added and were mixed
thoroughly by vortexing for 15 seconds till a
homogenous mixture was obtained. 400µl of 50%
ethanol was added and mixed by pipetting. The
prepared lysate was transferred to GeneJET
Genomic DNA Purification Column inserted in a
collection tube; the column was centrifuged at
6000xg for 60 seconds. After centrifugation flowthrough was discarded along with the collection
tube, the GeneJET Genomic DNA Purification
Column was placed into a new collection tube.
500µl of wash buffer I was added and centrifuged at
8000xg for 60 seconds, the flow through was
discarded and column was placed back into
collection tube and 500µl of wash buffer II was
added in each of tube and centrifuged at 12000xg
for 3minutes, the collection tube and flow-through
were discarded and column was placed in a new
sterilized 1.5ml eppendorf collection tube. 200µl of
elution buffer was added in each column in centre
of the GeneJET Genomic DNA Purification column
membrane to elute genomic DNA, columns were
incubated for 2 minutes at room temperature and
centrifuged for 1 minute at 8000xg. The purification
columns were discarded and the purified DNA was
immediately stored at -20°C after the measurement
of DNA concentration and absorption (purity) ratio.
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In another modified methods of above
commercial kit were used for the extraction of DNA
by increasing the 50% quantity of both reagents.
Chloroform - Phenol Method
Several chloroform phenol method were tested
for the extraction of DNA, after testing and making
several modifications in these methods, the
following method for extraction of DNA was
optimized which yielded good quality of DNA.
Each ovarian and testicle sample was added with 1
volume of Tris-saturated phenol and 1 volume of
chloroform and centrifuged at 10,000 rpm for 5
min, the upper aqueous phase of sample was
transferred in to the fresh tube and an equal volume
of chloroform was added, the sample was vortexed
and centrifuged at 10,000 rpm for 5 min at room
temperature, the aqueous phase was transferred in to
other fresh tube and added 1/10 the volume of 2 M
sodium chloride and 2.5 volumes of ethanol was
added and incubated the mixture for 30 min at 20°C followed by centrifugation at 10,000 rpm for
10 min. The supernatant was discarded and the
pellet was washed with 70% cold ethanol by
centrifugation at 10000 rom for 5/10 minutes, the
alcohol was completely discarded and allowed the
pellet to dry at room temperature. After complete
dryness the molecular grade water (nuclease-free)
was added and the measurement the purity of DNA
was recorded by Nanodrop.
Storage of Samples at Various Temperatures
After yielding of DNA from primary sex
organs (ovary and testes), the aliquotes of DNA
samples were stored at various temperatures i.e. 20°C, +4°C and at room temperature for 8 weeks.
The concentration and purity ratio of freshly
extracted and stored DNA at various temperatures
were
determined
by
Nanodrop
1000
spectrophotometer.
The obtained data was analyzed for ANOVA
and t test by statistical package (GraphPad Prism
and Insat 6).
Results
Storage of Extracted DNA from Ovaries at 20ºC, +4ºC and at Room Temperature
EFFECT OF STORAGE CONDITIONS ON DNA QUALITY OF PRIMARY SEX ORGANS …
When the extracted DNA sample from
modified method of kit and from chloroform was
stored at -20ºC temperature for 8 week it was found
that the DNA extracted from modified method of
normal kit was significantly (p=0.022) decreased
from 226.2 ng/μl to 109.7 ng/μl, similarly in
chloroform phenol method the quality of DNA was
degraded and measured concentration was 224.9
ng/μl than the normal concentration of 232.7 ng/μl
(Graph-1), however this difference was nonsignificant.
The DNA extracted from kit through modified
method and chloroform phenol methods was stored
at +4ºC for 8 week, the spectrophotometer
measured the lower concentration of DNA than the
fresh DNA (226.2 vs 107.1 ng/μl), and the DNA
extracted from chloroform and phenol method was
also degraded from 232.7 to 186.4 ng/μl (Graph-2).
When extracted DNA from modified methods
of kit and chloroform method was stored at room
temperature it was found that DNA was degraded in
both types of extractions. In modified method of kit
DNA was significantly (p=0.0003) degraded.
250
DNA (ng/μl)
200
150
233.4224.9
Fresh
100
50
226.2
109.7
-20°C
0
CK
PC
Extraction Methods
Graph-1: Comparison of concentration of ovarian DNA from fresh extraction and stored at -20°C.
p=0.022
CK= Commercial Kit.
PC= Phenol Chloroform.
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RANJHANI ET AL.
250
DNA (ng/ul)
200
150
232.7
100
226.2
186.4
Fresh
107.1
+4ºC
50
0
CK
PC
Extraction Methods
Graph-2: Comparison of concentration of ovarian DNA from fresh extraction and stored at +4°C.
CK= Commercial Kit.
PC= Phenol Chloroform.
250
DNA (ng/ul)
200
150
Fresh
100
226.2
92.2
232.7
50
101.6
Room Temp
0
CK
PC
Extraction Methods
Graph-3: Comparison of concentration of ovarian DNA from fresh extraction and stored at room temperature.
CK= Commercial Kit.
PC= Phenol Chloroform.
From 226.2 to 92.6 ng/μl and in chloroform
phenol method the stored DNA significantly
(p=0.028) decreased from 232.7 to 101.6 ng/μl
(Graph-3).
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J. Anim. Sci. Adv., 2014, 4(10): 1059-1067
Storage of DNA Extracted from Testes at 20ºC, +4ºC and Room Temperature
As shown in graph-4 the mean concentration of
DNA was degraded in both protocols of DNA
extraction. When compared with modified method
EFFECT OF STORAGE CONDITIONS ON DNA QUALITY OF PRIMARY SEX ORGANS …
of kit it was found that at -20°C the concentration
DNA was slightly degraded from 169.7 to 163.6
ng/µl, however, this difference was non-significant
and similar lower concentration from 233.4 to
229.4ng/µl was observed in the chloroform phenol
method.
The concentration of DNA was decreased in
both methods of extractions at +4ºC , in modified
kit method the mean concentration of testicle tissue
significantly (p=0.004) decreased from 169.7 to
107.1 ng/µl, also in chloroform phenol method the
DNA was degraded from 233.4 to 192.3 ng/µl
(Graph-5), however this difference was nonsignificant. The DNA stored at room temperature
for 8 weeks after the extraction, the concentration of
DNA was decreased in both methods of extractions,
in modified kit method the mean concentration of
testicle tissue significantly (p=0.0009) decreased
from 169.7 to 92.6 ng/µl, also in chloroform phenol
method the DNA was degraded from 233.4 to 111.3
ng/µl (Graph-6), this difference was statically nonsignificant.
250
DNA (ng/ul)
200
150
100
233.4
169.7
229.4
Fresh
163.6
-20ºC
50
0
CK
PC
Extraction Methods
Graph-4: Comparison of concentration of testicle DNA from fresh extraction and stored at -20°C.
CK= Commercial Kit.
PC= Phenol Chloroform.
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RANJHANI ET AL.
250
DNA (ng/μl)
200
150
Fresh
100
233.4
169.7
50
192.3
107.1
+4°C
0
CK
PC
Extraction Methods
Graph-5: Comparison of concentration of testicle DNA from fresh extraction and stored at +4°C.
CK= Commercial Kit.
PC= Phenol Chloroform.
250
DNA (ng/μl)
200
Fresh
150
100
169.7
92.6
233.4
111.3
Room
Temp
50
0
CK
PC
Extraction Methods
Graph-6: Comparison of concentration of testicle DNA from fresh extraction and stored at room temperature.
CK= Commercial Kit.
PC= Phenol Chloroform.
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EFFECT OF STORAGE CONDITIONS ON DNA QUALITY OF PRIMARY SEX ORGANS …
Discussion
The results of current study are in agreement
with Uren and Petal (2012) they found that
untreated samples produced a decrease in DNA
concentration at all analysis, similar results of the
DNA storage at room temperature for 21 months
could not amplify the target by (Colton and Clark
2001), similarly Lasken et al., (2003) stored DNA
samples at 4°C and room temperature showed
varying degrees of evaporation but DNA was stable
for up to 12 months at 4°C. The DNA was degraded
when Wan et al., (2010) evaluated the integrity and
quality of DNA stored for 3 weeks at room
temperature using Biometrical and GenVault
products against DNA stored at -20°C. When DNA
was stored for 2 years at room temperature there
was decrease in the number of observed alleles and
in the peak height of these alleles (Frippiat and Noel
2014). Recently, new room temperature storage
technologies tested on human DNA relying on
synthetic preservation matrices have been
developed (Hernandez et al., 2009; Wan et al.,
2010; Frippiat et al., 2011; Lee et al., 2012). This
result is in agreement with Howlett et al., (2014)
whose findings indicate that the -20°C controls and
the DNA stable protected samples at room
temperature provided similar DNA recoveries that
were higher compared to the unprotected controls
kept at room temperature, 37°C or 50°C, also the
DNA methylation is stable at room temperature
(Vilahur et al., 2013). DNA quality was best
preserved either dried or at -80°C; significant
quality loss occurred with -20°C and +4°C storage.
The similar results are reported by Smith and Morin
(2005) when samples were stored at -80°C,
regardless of storage additives, and those dried at
room temperature in the presence of trehalose.
Interestingly the sperm DNA from some sub
fertile stallions may decline at a greater rate than
spermatozoa from fertile stallions when exposed to
similar storage conditions (Lo et al., 2002), this
difference may be due to some morphological
changes in the spermatozoa.
In contrast to current results Wan et al., (2010)
stored DNA at room temperature for up to 3 weeks
and was equivalently to DNA kept frozen at -20°C
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with regard to downstream applications such as
short range PCR, long range PCR, DNA
sequencing, and SNP microarray analysis. This
difference may be due to the difference of
absorbance ratio of extracted DNA and residual of
nuclease remaining in the extraction, the different
protocol of DNA extraction and different incubation
period and type of tissue used. In conclusion
extraction from phenol chloroform method from
tissue of primary sex organs yielded better results
particularly when stored at -20°C compare to +4°C
and room temperature. The yielded DNA stored at 20°C can further be used for down streaming of
various molecular biological techniques like PCR,
gene sequencing, micro satellites etc.
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