an improved method for dna extraction from paraffin section yan-gao man, farid moinfar, gary l....
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An Improved Method An Improved Method for DNA Extraction for DNA Extraction
from Paraffin Sectionfrom Paraffin SectionYan-gao Man, Farid Moinfar, Gary Yan-gao Man, Farid Moinfar, Gary L. Bratthauer, Elizabeth A. Kuhls, L. Bratthauer, Elizabeth A. Kuhls,
Fattaneh A. TavassoliFattaneh A. Tavassoli
Department of Gynecologic and Breast Department of Gynecologic and Breast Pathology, Armed Forces Institute of Pathology, Armed Forces Institute of
PathologyPathology (( AFIPAFIP )) and American Registry of and American Registry of PathologyPathology (( ARPARP )) , Washington, DC 20306-, Washington, DC 20306-
6000, USA6000, USA
IntroductionIntroduction
Technical issues of DNA extractionTechnical issues of DNA extraction ::
◎ ◎ Evaluation of deparaffinizationEvaluation of deparaffinization
◎ ◎ Satisfied hematoxylin stainSatisfied hematoxylin stain
◎ ◎ Ratio of cell number to Ratio of cell number to enzyme volumeenzyme volume
◎ ◎ Monitor digestion processMonitor digestion process
IntroductionIntroduction
◎ ◎ ParaffinParaffin (( mp 40-60 ℃ mp 40-60 ℃ ))◎◎ Degree of freshness of xyleneDegree of freshness of xylene◎◎ TemperatureTemperature◎◎ DurationDuration◎◎ Thickness of sectionThickness of section
Deparaffinization Deparaffinization ::
IntroductionIntroduction
Hematoxylin stain Hematoxylin stain ::
IntroductionIntroduction
Hematoxylin stain Hematoxylin stain ::
IntroductionIntroduction
Hematoxylin stainHematoxylin stain ::
◎◎ Stable in dissection bufferStable in dissection buffer
◎◎ Non-lesion on re-cutsNon-lesion on re-cuts
◎◎ Tissue blocks not accessibleTissue blocks not accessible
◎◎ Reducing in 2Reducing in 2 % % hydrochloric acidhydrochloric acid
◎◎ Satisfied both morphological Satisfied both morphological and molecular assessmentsand molecular assessments
IntroductionIntroduction
Ratio of cell number to enzyme volume Ratio of cell number to enzyme volume ::
◎◎ Larger number of cellsLarger number of cells some cell undigested some cell undigested
◎◎ Larger enzyme volumeLarger enzyme volume low DNA content per unit low DNA content per unit
IntroductionIntroduction
◎◎ No practical mean to monitor No practical mean to monitor enzymatic digestion processenzymatic digestion process
Monitor digestion process Monitor digestion process ::
Materials and MethodMaterials and Method
◎◎ USUS 、、 JapanJapan 、、 ChinaChina 、、 AustriaAustria 、、ItalyItaly 、、 FranceFrance
◎◎ Age of the paraffin blocksAge of the paraffin blocks :: 3 3 months to over 30 yearsmonths to over 30 years
Slide prepare Slide prepare ::
NameName ManufactoryManufactory NoteNote
Microscope slidesMicroscope slides CMSCMS
Slide holdersSlide holders CMSCMS
Materials and MethodMaterials and Method
◎◎ ThicknessThickness :: 5-75-7μμmm (( a few at a few at 10-12 10-12 μμm m ))
◎◎ Clean cutting methodClean cutting method :: distilledistilled waterd water 、、 glovesgloves 、、 new bladenew blade
◎◎ Place vertically in 80℃ for 30-60 Place vertically in 80℃ for 30-60 minutesminutes
◎◎ Cool down 5-10 minutes at rooCool down 5-10 minutes at room temp.m temp.
Slide prepare Slide prepare ::
Materials and MethodMaterials and Method
NameName ManufactoryManufactory NoteNote
XyleneXylene Fisher ScientificFisher Scientific
EthanolEthanol Fisher ScientificFisher Scientific
Deparaffinization Deparaffinization ::◎ Xylene 3-5 minutes 3 times
◎ Descending concentration of ethanol 3-5 minutes
◎ Running tap water 5 minutes
Materials and MethodMaterials and Method
◎◎ Hematoxylin 30-60 secondsHematoxylin 30-60 seconds
◎◎ Rinse in tap waterRinse in tap water
StainStain ::
NameName ManufactoryManufactory NoteNote
HematoxylinHematoxylin Fisher ScientificFisher Scientific
Hydrochloric acidHydrochloric acid Fisher ScientificFisher Scientific
Ammonium Ammonium hydroxidehydroxide
LabChem IncLabChem Inc
GlycerinGlycerin CMSCMS
Materials and MethodMaterials and Method
◎ Dip in 2 % hydrochloric acid 3-5 times
◎ Running tap water 5-7 minutes
◎ 1X PBS ( pH7.4 ) contain 1 % ammonium hydroxide 5-7 minutes
StainStain ::
Materials and MethodMaterials and Method
◎◎ Evaluate the extent of Evaluate the extent of deparaffindeparaffin :: water retentionwater retention
hematoxylin stainhematoxylin stain
◎◎ Assess hematoxylin stainAssess hematoxylin stain :: nucleusnucleus
cytoplasmcytoplasm
◎◎ Distilled water 2-3 minutesDistilled water 2-3 minutes
◎◎ 1X PBS contain 101X PBS contain 10 % % glyceringlycerin
StainStain ::
Materials and MethodMaterials and Method
◎◎ DeparaffinDeparaffin
◎◎ Satisfactory stain for Satisfactory stain for morphologymorphology
◎◎ MicrodissectionMicrodissection
◎◎ 22 % % hydrochloric acid 3-5 hydrochloric acid 3-5 minutes 2 timesminutes 2 times
◎ ◎ 1X PBS1X PBS (( pH7.0pH7.0 )) 5-7 minutes5-7 minutes
◎ ◎ Centrifuge at 2000-3000g 3 minutesCentrifuge at 2000-3000g 3 minutes
◎ ◎ DigestionDigestion
A Different ApproachA Different Approach ::
Materials and MethodMaterials and Method
NameName ManufactoryManufactory NoteNote
MicrometerMicrometer Olympus Olympus OpticalOptical
30-Gauge needle30-Gauge needle Fisher Fisher ScientificScientific
Mineral oilMineral oil Fisher Fisher ScientificScientific
Microcentrifuge Microcentrifuge tubetube
MJ ResearchMJ Research
Proteinase KProteinase K SigmaSigma 10mg/10mg/
mlml ;; -80℃-80℃
Microdissection & enzymatic digestionMicrodissection & enzymatic digestion ::
Materials and MethodMaterials and Method
◎◎ MicrodissectionMicrodissection◎◎ Digestion bufferDigestion buffer :( :( fresh made fresh made ))
150 150 μμl proteinase K l proteinase K + + 850 850 μμl mixturel mixture
0.5 ml Tween 20 0.5 ml Tween 20 + 0.2 ml 0.5 M EDTA+ 0.2 ml 0.5 M EDTA (( pH8.0pH8.0 ))+ 5.0 ml 1 M Tris+ 5.0 ml 1 M Tris (( pH8.5pH8.5 ))+ 94.3 ml distilled water+ 94.3 ml distilled water
Microdissection & enzymatic digestionMicrodissection & enzymatic digestion ::
Materials and MethodMaterials and Method
◎◎ Add digestion bufferAdd digestion buffer
accord number of cellsaccord number of cells
◎◎ Mineral oilMineral oil
equal volume of digestion bufferequal volume of digestion buffer
◎◎ 37-40℃ 24-48 37-40℃ 24-48 hourshours (( magnifiermagnifier ))
◎◎ Incubate at 95℃ 10 minutesIncubate at 95℃ 10 minutes
◎ ◎ Store at 4℃Store at 4℃
Microdissection & enzymatic digestionMicrodissection & enzymatic digestion ::
Materials and MethodMaterials and MethodLoss of Heterozygosity Loss of Heterozygosity ::
NameName ManufactoryManufactory NoteNote
LOH markerLOH marker Research Research GeneticsGenetics
Gene Amp PCR kitGene Amp PCR kit Perkin-ElmerPerkin-Elmer
Taq gold DNA Taq gold DNA polymerasepolymerase Perkin-ElmerPerkin-Elmer
Gel loading bufferGel loading buffer Perkin-ElmerPerkin-Elmer
DNA size standardDNA size standard Perkin-ElmerPerkin-Elmer
Polyacryamide gelPolyacryamide gel Bio-RadBio-Rad
Materials and MethodMaterials and Method
◎◎ 30 fluorescent dye-labeled polymo30 fluorescent dye-labeled polymorphic DNA markers at 1rphic DNA markers at 1 、、 22 、、 33 、、1111 、、 1313 、、 16 and 17 16 and 17
◎◎ Annealing temperatureAnnealing temperature :: 55 - 6055 - 60℃℃
◎◎ 78 to 290 bp78 to 290 bp◎ ◎ Thermal cyclerThermal cycler :: Perkin-ElmerPerkin-Elmer
◎◎ 30 - 40 cycles30 - 40 cycles
Loss of Heterozygosity Loss of Heterozygosity ::
Materials and MethodMaterials and Method
◎ ◎ 5 – 6 5 – 6 % % Polyacryamide GelPolyacryamide Gel◎◎ Gel imageGel image :: Perkin-Elmer 377 DPerkin-Elmer 377 D
NA sequencerNA sequencer◎◎ LOHLOH :: 7575 % % reduction of one allreduction of one all
eleele
Loss of Heterozygosity Loss of Heterozygosity ::
Materials and MethodMaterials and Method
◎◎ DNA markers on exon 1 of the DNA markers on exon 1 of the human androgen receptor genehuman androgen receptor gene
◎◎ Hpa Hpa ⅡⅡ :: methylation sensitive methylation sensitive enzymeenzyme
◎ ◎ Rsa Rsa ⅠⅠ :: control enzymecontrol enzyme
◎ ◎ MonoclonalityMonoclonality :: One DNA band or One DNA band or peak after Hpa peak after Hpa ⅡⅡ digestion digestion
Clonality analysis Clonality analysis ::
NameName ManufactoryManufactory NoteNote
Hpa Hpa ⅡⅡ Gibco/BRL LifeGibco/BRL LifeRsa Rsa ⅠⅠ Gibco/BRL LifeGibco/BRL Life
Results and DiscussionResults and Discussion◎◎12 12 μμm thick human breastm thick human breast
No pretreate 80 30-60 minutes℃
1A放大 1C放大
Results and DiscussionResults and Discussion◎◎12 12 μμm thick human breastm thick human breast
No pretreate 80 30-60 minutes℃
Results and DiscussionResults and DiscussionNo pretreate 80 30-60 minutes℃
D16S518D3S1300D11S1311
160-160-300bp300bpTPO
106-106-130bp130bp
Results and DiscussionResults and Discussion
2 % hydrochloric acid
1 % ammonium hydroxide
Results and DiscussionResults and Discussion
◎ ◎ 10 10 μμl digestion bufferl digestion buffer :: 100-150 cells100-150 cells
◎ ◎ Monitoring the digestion process with a Monitoring the digestion process with a magnifiermagnifier
◎ ◎ >24 hours digestion>24 hours digestion
more concentrated enzyme solutionmore concentrated enzyme solution(( 10mg/ml10mg/ml )) re-deparaffinizationre-deparaffinization
Results and DiscussionResults and Discussion
Results and DiscussionResults and Discussion
Results and DiscussionResults and Discussion◎ ◎ Incubation of sections at 80℃ for 30-60 minutIncubation of sections at 80℃ for 30-60 minut
eses◎ ◎ 22 % % hydrochloric acid & 1hydrochloric acid & 1 % % ammonium hydammonium hyd
roxideroxide◎ ◎ 10 10 μμl l digestion bufferdigestion buffer :: 100-150 cells100-150 cells◎ ◎ Monitoring the digestion process with a magnMonitoring the digestion process with a magn
ifierifier◎ ◎ >24 hours digestion>24 hours digestion
more concentrated enzyme solutionmore concentrated enzyme solution (( 1010mg/mlmg/ml )) re-deparaffinizationre-deparaffinization
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