An Improved Method for DNA Extraction from Paraffin Section

1
An Improved Method for DNA Extraction from
Paraffin Section

• Yan-gao Man, Farid Moinfar, Gary L. Bratthauer,
  Elizabeth A. Kuhls, Fattaneh A. Tavassoli

Department of Gynecologic and Breast Pathology,
Armed Forces Institute of Pathology(AFIP)and
American Registry of Pathology(ARP), Washington,
DC 20306-6000, USA
2
Introduction

• Technical issues of DNA extraction

? Evaluation of deparaffinization ? Satisfied
hematoxylin stain ? Ratio of cell number to
enzyme volume ? Monitor digestion process
3
Introduction

• Deparaffinization

? Paraffin(mp 40-60 ? ) ? Degree of freshness of
xylene ? Temperature ? Duration ? Thickness of
section
4
Introduction

• Hematoxylin stain

5
Introduction

• Hematoxylin stain

6
Introduction

• Hematoxylin stain

? Stable in dissection buffer ? Non-lesion on
re-cuts ? Tissue blocks not accessible ? Reducing
in 2 hydrochloric acid ? Satisfied both
morphological and molecular assessments

7
Introduction

• Ratio of cell number to enzyme volume

? Larger number of cells ? some cell
undigested ? Larger enzyme volume ? low DNA
content per unit
8
Introduction
Monitor digestion process

• ? No practical mean to monitor enzymatic
  digestion process

9
Materials and Method
Slide prepare

• ? US?Japan?China?Austria?Italy?France
• ? Age of the paraffin blocks3 months to over 30
  years

Name Manufactory Note
Microscope slides CMS
Slide holders CMS
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Materials and Method
Slide prepare

• ? Thickness5-7µm(a few at 10-12 µm )
• ? Clean cutting methoddistilled water?gloves?new
  blade
• ? Place vertically in 80? for 30-60 minutes
• ? Cool down 5-10 minutes at room temp.

11
Materials and Method
Deparaffinization
? Xylene 3-5 minutes 3 times ? Descending
concentration of ethanol 3-5 minutes ? Running
tap water 5 minutes
Name Manufactory Note
Xylene Fisher Scientific
Ethanol Fisher Scientific
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Materials and Method
Stain

• ? Hematoxylin 30-60 seconds
• ? Rinse in tap water

Name Manufactory Note
Hematoxylin Fisher Scientific
Hydrochloric acid Fisher Scientific
Ammonium hydroxide LabChem Inc
Glycerin CMS
13
Materials and Method
Stain
? 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
14
Materials and Method
Stain

• ? Evaluate the extent of deparaffin
• ? water retention
• ? hematoxylin stain
• ? Assess hematoxylin stain
• ? nucleus
• ? cytoplasm
• ? Distilled water 2-3 minutes
• ? 1X PBS contain 10 glycerin

15
Materials and Method
A Different Approach

• ? Deparaffin
• ? Satisfactory stain for morphology
• ? Microdissection
• ? 2 hydrochloric acid 3-5 minutes 2 times
• ? 1X PBS(pH7.0)5-7 minutes
• ? Centrifuge at 2000-3000g 3 minutes
• ? Digestion

16
Materials and Method
Microdissection enzymatic digestion
Name Manufactory Note
Micrometer Olympus Optical
30-Gauge needle Fisher Scientific
Mineral oil Fisher Scientific
Microcentrifuge tube MJ Research
Proteinase K Sigma 10mg/ml-80?
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Materials and Method
Microdissection enzymatic digestion

• ? Microdissection
• ? Digestion buffer( fresh made )
• ? 150 µl proteinase K
• 850 µl mixture
• ? 0.5 ml Tween 20
• 0.2 ml 0.5 M EDTA(pH8.0)
• 5.0 ml 1 M Tris(pH8.5)
• 94.3 ml distilled water

18
Materials and Method
Microdissection enzymatic digestion

• ? Add digestion buffer
• ? accord number of cells
• ? Mineral oil
• ? equal volume of digestion buffer
• ? 37-40? 24-48 hours(magnifier)
• ? Incubate at 95? 10 minutes
• ? Store at 4?

19
Materials and Method
Loss of Heterozygosity
Name Manufactory Note
LOH marker Research Genetics
Gene Amp PCR kit Perkin-Elmer
Taq gold DNA polymerase Perkin-Elmer
Gel loading buffer Perkin-Elmer
DNA size standard Perkin-Elmer
Polyacryamide gel Bio-Rad
20
Materials and Method
Loss of Heterozygosity

• ? 30 fluorescent dye-labeled polymorphic DNA
  markers at 1?2?3?11?13?16 and 17
• ? Annealing temperature55 - 60?
• ? 78 to 290 bp
• ? Thermal cyclerPerkin-Elmer
• ? 30 - 40 cycles

21
Materials and Method
Loss of Heterozygosity

• ? 5 6 Polyacryamide Gel
• ? Gel imagePerkin-Elmer 377 DNA sequencer
• ? LOH75 reduction of one allele

22
Materials and Method
Clonality analysis

• ? DNA markers on exon 1 of the human androgen
  receptor gene
• ? Hpa ? methylation sensitive enzyme
• ? Rsa ?control enzyme
• ? MonoclonalityOne DNA band or peak after Hpa ?
  digestion

Name Manufactory Note
Hpa ? Gibco/BRL Life
Rsa ? Gibco/BRL Life
23
Results and Discussion
?12 µm thick human breast
No pretreate
80? 30-60 minutes
1A??
1C??
24
Results and Discussion
?12 µm thick human breast
No pretreate
80? 30-60 minutes
25
Results and Discussion
No pretreate
80? 30-60 minutes
D16S518 D3S1300 D11S1311 ?160-300bp TPO ?106-130
bp
26
Results and Discussion
1 ammonium hydroxide
2 hydrochloric acid
27
Results and Discussion

• ? 10 µl digestion buffer100-150 cells
• ? Monitoring the digestion process with a
  magnifier
• ? gt24 hours digestion
• ? more concentrated enzyme solution(10mg/ml)
• ? re-deparaffinization

28
Results and Discussion
29
Results and Discussion
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Results and Discussion

• ? Incubation of sections at 80? for 30-60 minutes
• ? 2 hydrochloric acid 1 ammonium hydroxide
• ? 10 µl digestion buffer100-150 cells
• ? Monitoring the digestion process with a
  magnifier
• ? gt24 hours digestion
• ? more concentrated enzyme solution(10mg/ml)
• ? re-deparaffinization

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THE END
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