rapd profiling, dna fragmentation, and …downloads.hindawi.com/journals/bmri/2017/8653286.pdf · 2...

Research ArticleRAPD Profiling DNA Fragmentationand Histomorphometric Examination inBrains of Wistar Rats Exposed to Indoor25Ghz Wi-Fi Devices Radiation

A O Ibitayo1 O B Afolabi2 A J Akinyemi2 T I Ojiezeh3

K O Adekoya4 and O O Ojewunmi5

1Biological Sciences Department Afe-Babalola University Ado-Ekiti Ekiti State Nigeria2Biochemistry Department Afe-Babalola University Ado-Ekiti Ekiti State Nigeria3Medical Laboratory Sciences Department Afe-Babalola University Ado-Ekiti Ekiti State Nigeria4Cell Biology and Genetics Department University of Lagos Akoka Lagos State Nigeria5DNA Laboratory National Sickle Cell Centre Idi Araba Lagos State Nigeria

Correspondence should be addressed to A O Ibitayo joke ibitayoyahoocom

Received 26 January 2017 Accepted 17 July 2017 Published 20 August 2017

Academic Editor Settimio Grimaldi

Copyright copy 2017 A O Ibitayo et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The advent of Wi-Fi connected high technology devices in executing day-to-day activities is fast evolving especially in developingcountries of the world and hence the need to assess its safety among others The present study was conducted to investigate theinjurious effect of radiofrequency emissions from installed Wi-Fi devices in brains of young male rats Animals were dividedinto four equal groups group 1 served as control while groups 2 3 and 4 were exposed to 25 Ghz at intervals of 30 45and 60 consecutive days with free access to food and water ad libitum Alterations in harvested brain tissues were confirmedby histopathological analyses which showed vascular congestion and DNA damage in the brain was assayed using agarose gelelectrophoresis Histomorphometry analyses of their brain tissues showed perivascular congestion and tissue damage as well

1 Introduction

Radiofrequency electromagnetic radiation (RF-EMR) emis-sion rates are fast increasing amongdeveloping countries overthe last decade This can be traced to the perceived need anduse of Internet services in executing daily activities at homework school and so forth [1] Technological advancementnecessitated the use ofWi-Fi connected devices in communi-cations research andmany other spheres of life and speedingup of day-to-day activities [2 3] The various sources of RF-EMF emission include devices such as phones and laptopsHowever a great debate exists about the possible damage theradiofrequency electromagnetic radiation (RF-EMR) emittedby mobile phones exerts on living systems which includesdamage to brain cells impairment of metabolic activities in

tissues and ultimately damage to the hereditary materialsDNA which serves as the underlying molecular chaperon ofdiverse metabolic activities that takes place in living systems[4ndash6] Health hazards from increased RF-EMF exposurehas been recognized to cause a wide range of biochemicaland physiological dysfunctions particularly in the brains ofyoung ones [7] Moreover the long-term RF-EMF exposuregenerates reactive oxygen species and different free radicals[8] Weak fields may speed up electron sharing process andthereafter weaken the H-bond of cellular macromoleculesThis phenomenon could describe the mechanism of tran-scription and protein translation which has been observedafter RF-EMF long-term exposure [9] On the other hand thetotal energy of weak EM-fields is not directly enough to breaka chemical bond in the genetic material [10] Consequently it

HindawiBioMed Research InternationalVolume 2017 Article ID 8653286 6 pageshttpsdoiorg10115520178653286

2 BioMed Research International

could be deduced indirectly that genotoxicity is arbitrated byindirectmechanisms and afterward the generation of reactiveradicals (ROS) or a disruption to DNA-repair processes[11 12] Also RF-EMF exposure can induce alteration inplasmamembrane potential and calcium effluxwith resultantcalcium diminution which leads to decrease in the activityof protein kinase C thereby promoting the alteration inmany enzymes ion pumps channels and proteins as wellas inducing apoptosis [13] Several studies confirmed thatexposure to electromagnetic fieldsmay increase the incidenceof cancer and DNA damage of sperm and brain cells [14]Other reports indicated deleterious hematopoietic indicesgastrointestinal reproductive effects and so forth [15ndash17]There are also experimental proofs of RF fields effects onhuman physiology and behavior at field strengths found inthe home or environment [18]

2 Materials and Methods

21 Animals and Experimental Design Sixteen adolescentWistar albino male rats weighing 80ndash120 g obtained from theanimal house of Faculty of Science Afe-Babalola UniversityNigeria were selected and used for the study They weremaintained for two weeks as acclimatization period understandard laboratory conditions with free access to food andwater provided ad libitum and subjected to natural lightfor 12 hours and dark for 12 hoursrsquo cycles After the periodof acclimatization rats were divided randomly into fourgroups 4 animals in each group The animals were pair-housed in groups of four in steel mesh cages and placedabout 10 cm away from installed devices that emitted RF-EMF The control group (119899 = 4) was kept in a steel meshcages with a glass barrier separating the control from theexperimental animalsThe radiation exposure on the animalswas varied from acute (30 days) subchronic (45 days) andchronic (60 days) exposure Upon arrival animals wereweighed and handled Ad libitum food (pelletized growersmash obtained from ABUAD farm) was weighed daily andgiven in equal proportions to the four groups Distilled waterwas provided in stainless steel containers for the duration ofthe experiment

22 Tissue Preparation At the end of exposure rats werefasted for 12 hours before being anesthetized and sacrificedby cervical dislocation They were decapitated and theirbrains were immediately harvested and washed using chilledsaline solution The harvested brains of the animals fromeach group were minced using a homogenizer and separatelyhomogenized (20wv) in ice coldTris-HCL (001M pH74)buffer

23 Histomorphometry Analysis Specimens of brain tis-sues were immediately fixed in 10 formal saline treatedwith conventional grade of alcohol and xylol embeddedin paraffin and sectioned at 4ndash6 120583 thickness The sectionswere stained with haematoxylin and eosin (HampE) stain forstudying the histomorphometry changes

Table 1 PCR primers used in RAPD-PCR

1 OPT ndash 06 - 51015840 ndash CAA GGG CAG A ndash 31015840

2 OPT ndash 07 - 51015840 ndash GGC AGG CTG T ndash 31015840

3 OPH ndash 09 - 51015840 ndash TGT AGC TGG G ndash 31015840

4 OPT ndash 12 51015840 ndash GCG TGT CTA G ndash 31015840

5 OPT ndash 20 51015840 ndash GAC CAA TGC C ndash 31015840

6 OPB ndash 10 51015840 ndash CTG CTG GGA C ndash 31015840

24 Random Amplified Polymorphic DNA (RAPD) Technique

241 Extraction of DNA DNA was extracted from brains ofthe four groups following the method described by Bardakciand Skibinski [19]

242 Polymerase Chain Reaction (PCR) Primers In thisstudy ten-base long oligonucleotides primers were usedto initiate the PCR amplifications Primers were randomlyselected based on GC content and annealing temperature forRAPD-PCR amplification (Table 1)

243 PCR Amplification and Agarose Gel ElectrophoresisPCR amplifications were performed as described byWilliamset al 1993 [20] using the isolated DNA from one sample ofeach group

244 Agarose Gel Electrophoresis The amplified DNA frag-ments were separated on 15 agarose gel and stainedwith ethidium bromide DNA ladder 1 kb plus ladder fromThermos Scientific was used in this study as marker foramplified pattern The amplified pattern was visualized andphotographed by gel documentation system

25 Statistical Analysis The data entry was done into abinary data matrix as discrete variables and was analyzedaccording to Steel and Torrie [21] Statistical significanceof the difference in values of control and treated animalswas calculated at 5 significance level Data of the presentstudy were statistically analyzed by using Duncanrsquos multiplerange test (SAS Analytics) All RAPD profiles were analyzedusing stat program which showed the similarity between theamplified PCR products The best amplified PCR productof each group was selected to compare between the geneticlevels

3 Results

31 DNA Fragmentation and Genetic Analysis Using RAPD-PCR Six of 10-mer primers were used for investigating thesignificant changes of the DNA isolated from brain tissueswhich was loaded on a 15 agarose gel Two out of the six10-mer primers (OPT ndash 20 and OPB ndash 10) produced clearsharp monomorphic and polymorphic bands as seen inFigure 1 Primers 1 and 4 (OPT 06 and OPT 12) gave bandpatterns of almost the same profile between the four as seen inFigure 2 although some bands were lost In contrast primer2 and primer 3 (OPT 07 and OPH 09) were least informative

BioMed Research International 3

3000

1500

900

700

600

500

400

300

200

(Primers) OPT-20 OPB-10

grp2 grp4grp1 grp31 kbplus grp2 grp4grp1 grp31 kbplus

Figure 1 Figure 1 shows the RAPD profile of group 1 control (noradiofrequency exposure) group 2 (30-day RF exposure) group 3(45-day RF exposure) and group 4 (60-day RF exposure) animalsusing OPH-20 and OPB-10 primer

30001500

900700600500400300200

(Primers) T-06T-12


Figure 2 RAPD profile of group 1 control (no radiofrequencyexposure) group 2 (4-week RF exposure) group 3 (6-week RFexposure) and group 4 (8-week RF exposure) animals using T-12and T-06 primers

and they producedmostly undistinguishable banding profilesbetween the amplified samples of each group after RAPDassays as shown in Figure 3 The amplified fragments ofPCR products were summarized as in Table 2 Primers 1 and6 produced almost similar RAPD fingerprints for controlgroup (group 1) while primers 1 4 and 6 detected somechanges in brain DNA of subchronic (group 3 for 45 days)and chronic (group 4 for 60 days) group showing similarband sizes Primers 2 and 3 produced almost similar RAPDfingerprints for subchronic (group 3 for 45 days) and chronic(group 4 for 60 days) group although some bands were lostThe RAPD products were scored as present (1) or absent(0) for each primer-genotype combination The results ofRAPD patterns of the 6 primers were summarized as inTable 2 Sixteen bands were amplified and scored where4 were polymorphic Jaccardrsquos coefficient of similarity wasmeasured and a dendrogram constructed based on similaritycoefficients which was generated by using unweighted pairgroup method with arithmetic mean (UPGMA) The bestamplified PCR was selected from each group to compare

30001500

900700600500400300200

(Primers) T-09T-07



Grp1 (control) Grp2 Grp3 Grp4

Figure 4 DNA fragmentation pattern using agarose gel elec-trophoresis

between them using stat software The analysis of the resultsdescribed the similarity between different samples of braintissues The similarity of control (group 1) and group 2 (acuteexposure) was about 61 and 71 respectively comparedto group 3 and 4 The variations of the RAPD profiles werecompared between the control and Wi-Fi-exposed groups

We also observed a progressive DNA fragmentation inthe band pattern as shown in Figure 4 using agarose gelelectrophoresis where the DNA from the brains of thecontrol rats (group 1) and group 2 (30-day RF exposurespectrum) showed similar fragmentation pattern and largerband sizes while the bands of groups 3 (45-day RF exposurespectrum) and 4 (60-day RF exposure spectrum) indicatedDNAdamage and fragmented band sizes signifying apoptosisfrom subchronic and chronic Wi-Fi exposed groups

32 Histomorphometric Examinations The histomorphome-tric studies of the groups are shown in Figure 5

4 Discussion

Radiation has been speculated to be an environmental pol-lutant and its toxicity has also been associated with health


Table 2

Primer Number of amplified DNA fragment Number of polymorphic DNA fragment polymorphism indexingOPH-09 3 1 333OPT-06 2 mdash mdashOPT-07 3 3 100OPB-10 3 mdash mdashOPT-12 1 mdash mdashOPT-20 4 mdash mdashTotal 16 4 mdash 25

Control

Group 1

Group 2

Group 3

000 025 050 075 100

Coefficient

problems The brain is the closest organ in the body to theear when calls are received or placed Hence it is one ofthe target organs affected by radiation exposure owing to itsfluidity and ions retention especially in young children [722] This accounts for the restrictions and recommendationson the bandwidth rate and types of cell phones that canbe used by children in some countries Data shown inTable 2 demonstrated that subchronic and chronic exposureto Wi-Fi radiation indicated DNA damage in their braintissues while acute exposure revealed also relative effectswhich were not as severe as the longer exposure periodsAlso the PCR product using selected RAPD-PCR markerassisted analysis gave an evidence of DNA damage andmutations which was reflected in their RAPD profiles suchas disappearance of bands and appearance of new PCRproducts which occurred in the profiles generated by ratsexposed to Wi-Fi radiation RAPD-PCR was used here toanalyze induced DNA damage and changes in band patternsobserved in DNA ldquofingerprintrdquo analyses which reflects DNAalterations from single base changes (point mutations) tocomplex chromosomal rearrangements Furthermore ran-dom amplified polymorphism of DNA (RAPD) showeddistinct differences in animal groups exposed for acute (30

days) subchronic (45 days) and chronic exposure periodsAlso gel electrophoresis of extracted DNA samples fromthese groups revealed a dose-dependent (based on exposureduration) deleterious effect of Wi-Fi radiation on the braintissues and cells as shown in Figure 4 In addition to thisFigures 5(b) and 5(d) showed that Wi-Fi radiation exposureinduced severe histopathological alterations in the acuteand chronically exposed animals when compared to thecontrol animals where there was vascular congestion andabnormal architecture which is indicative of oxidative stressand neuroinflammation and this is in consonance with thefindings of [12 18 22] although radiation effects as indicatedin animals from the subchronic group did not really exhibitsevere histopathological changes as being observed in theacute and chronically exposed groups

5 Conclusion

In this study the effect of Wi-Fi radiation exposure as athreat to brain health was studied using genomic analysisand histopathological study which showed the high riskof its genotoxicity especially in prolonged exposure spec-trum through the findings from this study The genomic


(a) (b)

(c) (d)

Figure 5 Paraffin sections stained by haematoxylin and eosin (HampE) times400 for histomorphometry examination of brain (cerebrum) tissues ofrats as follows (a) Photomicrograph of control group shows normal architecture (H) section of the brain is composed of nerve cells (NC) andglia cells (arrow) and blood vessel (arrow head) disposed within the brain parenchyma Section is free from collections and inflammatory cellsinfiltrations (b) Photomicrograph of group 2 (30-day RF exposure) shows section of the brain with vascular congestion (L) the parenchymais free from collection (c) Photomicrograph of group 3 (45-day RF exposure) shows sections-nerve cells (NC) and neuroglia (arrow) appearas in control (d) Photomicrograph of group 4 (chronic exposure) shows section of the brain with vascular congestion (L) the parenchymais free from collection

analysis confirmed DNA damage due to Wi-Fi radiationtoxicity and DNA damage effect which was seen throughthe RAPD profiles of animals from the exposed groupsThe histopathological analyses also confirmed significantdeleterious alterations in the brain tissues of Wi-Fi-exposedanimals Hence the need to exhibit caution in handling smartdevices that are used from day to day is fast becoming a threatto human health and wellness

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

References

[1] L Hardell and C Sage ldquoBiological effects from electromagneticfield exposure and public exposure standardsrdquo Biomedicine andPharmacotherapy vol 62 no 2 pp 104ndash109 2008

[2] OB Afolabi and AO Ibitayo ldquoFadaka AOOverview of chromo-somal damage in abnormal absorption of magnetic and radio

waves Implications among cell-phone userrdquo Pharmacologyon-line p 3 2015

[3] A Abdolmaleki F Sanginabadi and A Rajabi ldquoSaberiRTheeffect of EMF wave exposure on blood parametersrdquo Int JHeamatOncol Stem Cell Res vol 6 no 2 pp 13ndash16 2012

[4] S Shokri A Soltani M Kazemi and D Sardari ldquoBaba-poorFMEffects of Wi-Fi9245GHz) exposure on Apoptosisspermparametres and testicular histomorphometry in rats Atime course studyrdquo Cell Journal vol 17 no 2 pp 322ndash331 2015

[5] C M Chaturvedi V P Singh P Singh et al ldquo245GHz (CW)Microwave irradiation alters circadian organization spatialmemory DNA structure in the brain cells and blood cell countsof male mice mus musculusrdquo Progress In ElectromagneticsResearch B no 29 pp 23ndash42 2011

[6] K K Kesari S Kumar and J Behari ldquoEffects of radiofrequencyelectromagnetic wave exposure from cellular phones on thereproductive pattern in male Wistar ratsrdquo Applied Biochemistryand Biotechnology vol 164 no 4 pp 546ndash559 2011

[7] C Marino I Lagroye M R Scarfı and Z Sienkiewicz ldquoArethe young more sensitive than adults to the effects of radiofre-quency fields An examination of relevant data from cellularand animal studiesrdquo Progress in Biophysics and MolecularBiology vol 107 no 3 pp 374ndash385 2011


[8] M Zmyslony P Politanski E Rajkowska W Szymczak andJ Jajte ldquoAcute exposure to 930 MHz CW electromagneticradiation in vitro affects reactive oxygen species level in ratlymphocytes treated by iron ionsrdquo Bioelectromagnetics vol 25no 5 pp 324ndash328 2004

[9] A Maziarz B Kocan M Bester et al ldquoHow electromagneticfields can influence adult stem cells Positive and negativeimpactsrdquo Stem Cell Research and Therapy vol 7 no 1 articleno 54 2016

[10] K R Foster ldquoRadiofrequency exposure from wireless lansutilizing Wi-Fi technologyrdquo Health Physics vol 92 no 3 pp280ndash289 2007

[11] F Ozguner A Altinbas M Ozaydin et al ldquoMobile phone-induced myocardial oxidative stress protection by a novelantioxidant agent caffeic acid phenethyl esterrdquo Toxicology andIndustrial Health vol 21 no 9 pp 223ndash230 2005

[12] M Naziroglu M Yuksel S A Kose and M O OzkayaldquoRecent reports of Wi-Fi and mobile phone-induced radiationon oxidative stress and reproductive signaling pathways infemales and malesrdquo Journal of Membrane Biology vol 246 no12 pp 869ndash875 2013

[13] O Johansson ldquoDisturbance of the immune system by elec-tromagnetic fieldsmdasha potentially underlying cause for cellulardamage and tissue repair reduction which could lead to diseaseand impairmentrdquo Pathophysiology vol 16 no 2-3 pp 157ndash1772009

[14] Vijayalaxmi and T J Prihoda ldquoGenetic damage in mammaliansomatic cells exposed to radiofrequency radiation A meta-analysis of data from 63 publications (1990-2005)rdquo RadiationResearch vol 169 no 5 pp 561ndash574 2008

[15] R Van Den Heuvel H Leppens G Nemethova and LVerschaeve ldquoHaemopoietic cell proliferation in murine bonemarrow cells exposed to extreme low frequency (ELF) electro-magnetic fieldsrdquoToxicology in Vitro vol 15 no 4-5 pp 351ndash3552001

[16] V S Benson K Pirie G K Reeves Gillian K V Beral J Greenand J Schuz ldquoMobile phone use and risk of brain neoplasmsand other cancers Prospective studyrdquo International Journal ofEpidemiology vol 42 no 3 pp 792ndash802 2013

[17] A Agarwal F Deepinder R K Sharma G Ranga and J LildquoEffect of cell phone usage on semen analysis in men attendinginfertility clinic an observational studyrdquo Fertility and Sterilityvol 89 no 1 pp 124ndash128 2008

[18] J Miyakoshi ldquoCellular and molecular responses to radio-frequency electromagnetic fieldsrdquo Proceedings of the IEEE vol101 no 6 pp 1494ndash1502 2013

[19] F Bardakci and D O F Skibinski ldquoApplication of the RAPDtechnique in tilapia fish species and subspecies identificationrdquoHeredity vol 73 no 2 pp 117ndash123 1994

[20] J G KWilliams M K Hanafey J A Rafalski and S V TingeyldquoGenetic analysis using random amplified polymorphic DNAmarkersrdquoMethods in Enzymology vol 218 pp 704ndash740 1993

[21] DG Steel and JH TorriePrinciples andProcedures of StatisticsMcGraw-Hill BookCompanyNewYorkNYUSA 2nd edition1981

[22] I Yakymenko E Sidorik S Kyrylenko andV Chekhun ldquoLong-term exposure to microwave radiation provokes cancer growthEvidences from radars and mobile communication systemsrdquoExperimental Oncology vol 33 no 2 pp 62ndash70 2011

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


could be deduced indirectly that genotoxicity is arbitrated byindirectmechanisms and afterward the generation of reactiveradicals (ROS) or a disruption to DNA-repair processes[11 12] Also RF-EMF exposure can induce alteration inplasmamembrane potential and calcium effluxwith resultantcalcium diminution which leads to decrease in the activityof protein kinase C thereby promoting the alteration inmany enzymes ion pumps channels and proteins as wellas inducing apoptosis [13] Several studies confirmed thatexposure to electromagnetic fieldsmay increase the incidenceof cancer and DNA damage of sperm and brain cells [14]Other reports indicated deleterious hematopoietic indicesgastrointestinal reproductive effects and so forth [15ndash17]There are also experimental proofs of RF fields effects onhuman physiology and behavior at field strengths found inthe home or environment [18]

2 Materials and Methods

21 Animals and Experimental Design Sixteen adolescentWistar albino male rats weighing 80ndash120 g obtained from theanimal house of Faculty of Science Afe-Babalola UniversityNigeria were selected and used for the study They weremaintained for two weeks as acclimatization period understandard laboratory conditions with free access to food andwater provided ad libitum and subjected to natural lightfor 12 hours and dark for 12 hoursrsquo cycles After the periodof acclimatization rats were divided randomly into fourgroups 4 animals in each group The animals were pair-housed in groups of four in steel mesh cages and placedabout 10 cm away from installed devices that emitted RF-EMF The control group (119899 = 4) was kept in a steel meshcages with a glass barrier separating the control from theexperimental animalsThe radiation exposure on the animalswas varied from acute (30 days) subchronic (45 days) andchronic (60 days) exposure Upon arrival animals wereweighed and handled Ad libitum food (pelletized growersmash obtained from ABUAD farm) was weighed daily andgiven in equal proportions to the four groups Distilled waterwas provided in stainless steel containers for the duration ofthe experiment

22 Tissue Preparation At the end of exposure rats werefasted for 12 hours before being anesthetized and sacrificedby cervical dislocation They were decapitated and theirbrains were immediately harvested and washed using chilledsaline solution The harvested brains of the animals fromeach group were minced using a homogenizer and separatelyhomogenized (20wv) in ice coldTris-HCL (001M pH74)buffer

23 Histomorphometry Analysis Specimens of brain tis-sues were immediately fixed in 10 formal saline treatedwith conventional grade of alcohol and xylol embeddedin paraffin and sectioned at 4ndash6 120583 thickness The sectionswere stained with haematoxylin and eosin (HampE) stain forstudying the histomorphometry changes

Table 1 PCR primers used in RAPD-PCR

1 OPT ndash 06 - 51015840 ndash CAA GGG CAG A ndash 31015840

2 OPT ndash 07 - 51015840 ndash GGC AGG CTG T ndash 31015840

3 OPH ndash 09 - 51015840 ndash TGT AGC TGG G ndash 31015840

4 OPT ndash 12 51015840 ndash GCG TGT CTA G ndash 31015840

5 OPT ndash 20 51015840 ndash GAC CAA TGC C ndash 31015840

6 OPB ndash 10 51015840 ndash CTG CTG GGA C ndash 31015840

24 Random Amplified Polymorphic DNA (RAPD) Technique

241 Extraction of DNA DNA was extracted from brains ofthe four groups following the method described by Bardakciand Skibinski [19]

242 Polymerase Chain Reaction (PCR) Primers In thisstudy ten-base long oligonucleotides primers were usedto initiate the PCR amplifications Primers were randomlyselected based on GC content and annealing temperature forRAPD-PCR amplification (Table 1)

243 PCR Amplification and Agarose Gel ElectrophoresisPCR amplifications were performed as described byWilliamset al 1993 [20] using the isolated DNA from one sample ofeach group

244 Agarose Gel Electrophoresis The amplified DNA frag-ments were separated on 15 agarose gel and stainedwith ethidium bromide DNA ladder 1 kb plus ladder fromThermos Scientific was used in this study as marker foramplified pattern The amplified pattern was visualized andphotographed by gel documentation system

25 Statistical Analysis The data entry was done into abinary data matrix as discrete variables and was analyzedaccording to Steel and Torrie [21] Statistical significanceof the difference in values of control and treated animalswas calculated at 5 significance level Data of the presentstudy were statistically analyzed by using Duncanrsquos multiplerange test (SAS Analytics) All RAPD profiles were analyzedusing stat program which showed the similarity between theamplified PCR products The best amplified PCR productof each group was selected to compare between the geneticlevels

3 Results

31 DNA Fragmentation and Genetic Analysis Using RAPD-PCR Six of 10-mer primers were used for investigating thesignificant changes of the DNA isolated from brain tissueswhich was loaded on a 15 agarose gel Two out of the six10-mer primers (OPT ndash 20 and OPB ndash 10) produced clearsharp monomorphic and polymorphic bands as seen inFigure 1 Primers 1 and 4 (OPT 06 and OPT 12) gave bandpatterns of almost the same profile between the four as seen inFigure 2 although some bands were lost In contrast primer2 and primer 3 (OPT 07 and OPH 09) were least informative


3000

1500

900

700

600

500

400

300

200




30001500

900700600500400300200

(Primers) T-06T-12




30001500

900700600500400300200

(Primers) T-09T-07








4 Discussion



Table 2


Control

Group 1

Group 2

Group 3

000 025 050 075 100

Coefficient



5 Conclusion



(a) (b)

(c) (d)





References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















3000

1500

900

700

600

500

400

300

200




30001500

900700600500400300200

(Primers) T-06T-12




30001500

900700600500400300200

(Primers) T-09T-07








4 Discussion



Table 2


Control

Group 1

Group 2

Group 3

000 025 050 075 100

Coefficient



5 Conclusion



(a) (b)

(c) (d)





References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 2


Control

Group 1

Group 2

Group 3

000 025 050 075 100

Coefficient



5 Conclusion



(a) (b)

(c) (d)





References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)





References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















rapd profiling, dna fragmentation, and …downloads.hindawi.com/journals/bmri/2017/8653286.pdf · 2...

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