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 ◎ No practical mean to monitor enzymatic digestion process Monitor digestion process ：

9. Materials and Method Slide prepare ： ◎ US、Japan、China、Austria、Italy、France ◎ Age of the paraffin blocks：3 months to over 30 years

10. Materials and Method Slide prepare ： ◎ Thickness：5-7μm（a few at 10-12 μm ） ◎Clean cutting method：distilled 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

12. Materials and Method Stain： ◎ Hematoxylin 30-60 seconds ◎ Rinse in tap water

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：

17. 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 ：

20. Materials and Method Loss of Heterozygosity ： ◎ 30 fluorescent dye-labeled polymorphic DNA markers at 1、2、3、11、13、16 and 17 ◎ Annealing temperature：55 - 60℃ ◎ 78 to 290 bp ◎ Thermal cycler：Perkin-Elmer ◎ 30 - 40 cycles

21. Materials and Method Loss of Heterozygosity ： ◎ 5 – 6 ％ Polyacryamide Gel ◎ Gel image：Perkin-Elmer 377 DNA sequencer ◎ LOH：75％ reduction of one allele

22. Materials and Method Clonality analysis ： ◎ DNA markers on exon 1 of the human androgen receptor gene ◎ Hpa Ⅱ：methylation sensitive enzyme ◎ Rsa Ⅰ：control enzyme ◎ Monoclonality：One DNA band or peak after Hpa Ⅱ digestion

23. Results and Discussion ◎12 μm thick human breast No pretreate 80℃ 30-60 minutes 1A放大 1C放大

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 106-130bp

26. Results and Discussion 1％ ammonium hydroxide 2％ hydrochloric acid

27. Results and Discussion ◎ 10 μl digestion buffer：100-150 cells ◎ Monitoring the digestion process with a magnifier ◎ >24 hours digestion  more concentrated enzyme solution（10mg/ml）  re-deparaffinization

28. Results and Discussion

29. Results and Discussion

30. Results and Discussion ◎ Incubation of sections at 80℃ for 30-60 minutes ◎ 2％ hydrochloric acid & 1％ ammonium hydroxide ◎ 10 μl digestion buffer：100-150 cells ◎ Monitoring the digestion process with a magnifier ◎ >24 hours digestion  more concentrated enzyme solution（10mg/ml）  re-deparaffinization

31. THE END

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