an improved method for dna extraction from paraffin section yan-gao man, farid moinfar, gary l....

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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