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Expression patterns and copy number changes in DFSP  
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Materials and Methods
 
DNA isolation from frozen or formalin fixed, paraffin embedded tissue.
Protocol last updated: February 28, 2003
1. DNA isolation from frozen tissue homogenized in TRIzol
To be prepared:

Back extraction buffer (BEB)

For 250 ml BEB (take precautions --toxic substances--):

Take 150 ml of millipore water and dissolve:
  • - 118.2 g of Guanidine Thiocyanate (FW 118.2) = 4 M
  • - 3.68 g of Sodium Citrate NaCi (FW 294.1) = 50 mM
  • - 30.29 g of Tris (free base)Tris (FW 121.14) = 1M
  • - add additional millipore water until volume is 250 ml
  • - sterilize bottle


  • 3 M Sodium acetate pH 5.2-7

    1 x TE buffer pH 8.0

    phenol/chloroform/isoamylalcohol 25:24:1 (PCI) (toxic!!)


    1. Homogenize tissue in TRIzol (Invitrogen) and incubate for 5-10 minutes at room temperature (RT). 0.2-1 gram of tissue can be placed in 10-12 ml of TRIzol. For less weight, apply the rule of (at least) 1 ml TRIzol per 50-100 mg of tissue processed. For details see RNA isolation protocol.
    2. Add 0.2 ml chloroform per 1 ml TRIzol reagent used in step 1. Shake tube vigorously for 5 minutes at RT. Incubate another 5-10 minutes on a shaking device. (Omit step 2 of the RNA isolation protocol to get rid of cell debris by spinning this down).
    3. Spin at 12,000 x g for 15 min. at 4° C. Separate the upper aqueous phase carefully, which contains the total RNA, and place this in a new tube. For total RNA isolation, see above mentioned protocol.
    4. Spin down the tubes which contain the interphase/organic phase with TRIzol at 12,000 x g for 5 min. at 4° C. Remove any remaining aqueous phase carefully which would contaminate your DNA sample with RNA. Now the interphase contains your DNA. At this point, you can store your samples at 4° C for days to weeks.
    5. Add 0.25-5 ml of the BEB (back extraction buffer) per 1 ml of TRIzol used for the RNA extraction to each tube. Mix intensively at least 3 min. by inversion or put on a shaker for 10 min.
    6. Spin tubes at 12,000 x g for 30 minutes at room temperature.
    7. Transfer upper aqueous phase and eventually save interphase for protein isolation.
    8. Add 0.4 ml isopropanol per 1 ml TRIzol used for original RNA isolation. Mix and incubate for 5 min. at RT.
    9. Spin samples at 12,000 x g for 15 min. at 4° C.
    10. Remove supernatant, keep pellet with DNA. Add 0.5 ml of ethanol 70% per 1 ml TRIzol used for RNA extraction and wash pellet by inversion.
    11. Spin down at 12,000 g for 15 min. at 4° C. Remove ethanol and dissolve pellet in about 400 ul of 1x TE buffer. DNA can be stored at 4° C. This DNA will perform fine in many reactions.

      For CGH additional washing steps with phenol, chloroform and isoamylalcohol are recommended:

    12. For these steps the use of Phase Lock Gel (PLG) tubes (light 2.0 ml, Eppendorf) are recommended. Spin down PLG tubes at 12,000-16,000 x g in microcentrifuge for 20-30 sec.
    13. Add 400 ml of DNA in 1xTE buffer pH 8.0 and add equal volume of PCI directly to the tube. Mix the organic and aqueous phases intensively by inverting and shake 10 min.
    14. Centrifuge at 10,000-12,000 x g for 15 min. at RT to separate the phases. The PCI will be located at the bottom of the tube, while the dissolved DNA will remain above the gel.
    15. Add the same volume of PCI as in step 13 and mix and centrifuge again (step 14).
    16. take new tubes, transfer aqueous phase to a new tube, add the same volume ( about 200 ml) of chloroform-isoamylalcohol (or chloroform only) to the upper aqueous phase and centrifuge as in step 14.
    17. Transfer the upper aqueous phase to a new 1.5 - 2.0 ml microfuge tube.
    18. Add 20 ml (10% of the volume) of 3M Sodium Acetate pH 5.2-7.0. Mix and add 2 to 2.5 volumes of 95-100% of ethanol. Mix and you can see the DNA precipitate.
    19. Spin down the DNA for 10-15 minutes in a micro-centrifuge at full speed.
    20. Discard the liquid and add 100 ml of ice cold 70% ethanol. Spin down DNA for 10-15 minutes in a micro-centrifuge at full speed.
    21. Discard the liquid, use pipette to evacuate last bit of fluid, make sure no liquid is left in the tube. Resuspend pellet in 1 x TE or DNAse free water, let DNA stay at RT for 5-6 hours or overnight.
    22. Measure OD and run 1% agarose gel to check DNA quality. Store DNA at 4° C until use.

    2. DNA isolation from formalin-fixed, paraffin-embedded tissue

    1. Cut three to six 20 um sections and place them into a 1.5 ml micro centrifuge tube. The total amount of sections depends on tissue size, type and age of your samples. Weigh the total amount of tissue obtained.
    2. Add 1 ml of xylene. Rock at RT for 5 min. Centrifuge at full speed for 3 min.
    3. Remove supernatant by pipetting. Do not remove any of the pellets.
    4. Repeat steps 2-3, 3-4 times (at least 3 xylene washes).
    5. Add 1 ml of 100% ethanol. Rock at RT for 5 min. Centrifuge at full speed for 3 min.
    6. Remove supernatant by pipetting. Do not remove any of the pellets.
    7. Repeat steps 5-6, 3-4 times (at least 3 ETOH washes).
    8. Vacuum-dry (without heat) or air-dry pellet completely.

      From here follow the protocol of QIAamp DNA Mini Kit (QIAGEN Cat No.: 51304) with some modifications (all steps at RT, unless stated otherwise).


    9. Resuspend the tissue pellet in 180 ul Buffer ATL (QIAamp DNA Mini Kit).
    10. Mix well, let it sit at RT for about 30 min.
    11. Add 20-30 ul Proteinase K (provide from the Kit), mix by vortexing, and incubate at 56° C in a shaking water bath overnight or until tissue is completely lysed.
    12. Spin down at maximum speed for 5-10 min, and transfer the supernatant into a new tube.
    13. Add 4 ul of RNase A (100mg/ml)(QIAGEN, Cat. No. 19101), mix by pulse-vortexing for 15 seconds, incubate for 2 min. at room temperature.
    14. Briefly spin, then add 200 ul AL Buffer (QIAamp DNA Mini Kit), mix again by pulse-vortexing for 15 seconds. It is essential that the sample and AL Buffer are mixed thoroughly to yield a homogeneous solution.
    15. Incubate at 70°C for 10 min, briefly spin to remove drops from the inside of lid. In most cases, a white precipitation will be dissolved during incubation.
    16. Add 200 ul of 100% ethanol, mix by pulse-vortexing for 15 seconds, briefly spin. It is essential that the sample, buffer AL, and the ethanol are mixed thoroughly to yield a homogeneous solution. For optimal binding conditions, it might help to increase the volume of ethanol from 200 ul to 230 ul. If you started with a sample of more than 25 mg, increase the amount of ethanol proportionally; a 50 mg (360 ul of ATL) sample will require 400-460 ul ethanol.
    17. Add all of the solution including the precipitate to the QIAamp spin column.
    18. Incubate at RT for 5 min. Spin at 6,000 x g for 1 min.
    19. Reload the flow-thru to the spin column again.
    20. Repeat step 18 and 19 1-2 times. This may increase the yield.
    21. Place the QIAamp spin column in a clean 2 ml collection tube; discard the tube containing the filtrate.
    22. Add 500 ul Buffer AW1 (QIAamp DNA Mini Kit, washing buffer), spin at same speed as above for 1 min.
    23. Add 500ul Buffer AW2 (QIAamp DNA Mini Kit, washing buffer), spin at maximum speed (20,000 x g) for 3 min.
    24. Place the QIAamp column in a new 2 ml collection tube, spin at maximum speed for 1 min. This is because residual Buffer AW2 in the elution may cause problems in further applications.
    25. Place QIAamp column in a clean 1.5 ml tube, add 50-100 ul Buffer AE or distilled water. Optional: Use AE or distilled water at 55°-65° C. To increase the final DNA concentration one can use a volume of 30 to 50 ul.*
    26. Incubate at room temperature for 5 min.
    27. Spin at 6000 x g for 1 min.
    28. Save the elution, this is your DNA.
    29. Repeat steps 28-31.
    30. Pool the 2 x 50-100 ul of DNA.
    31. Measure OD by spectophotometry. Purity is determined by A260/A280 ratio (1.6 - 1.7). Check the quality and quantity of the DNA by running on a 1% agarose gel. DNA size is mostly about 500 bp - 3,000 bp or up with a smear on a 1% agarose gel. Some DNAs showed bands of up to 10,000 bp.
    32. Store the sample at 4°C or -20° C for long term until use.
  • * Yields will depend both on the size and the age of the sample processed. Reduced yields compared to fresh or frozen tissue are expected. Therefore, eluting the DNA in 50-100 ul or less with buffer AE or distilled H2O is recommended.

  • * 1 mg of frozen tissue will yield approximately 0.2-1.2 ug of DNA.
  • For any questions, remarks, please feel free to contact (see author list for details)

    Matt van de Rijn, MD, PhD
    Sabine Linn, MD, PhD


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