DNA Extraction from Paraffin Embedded Tissues

Gregory Buzard buzardg at MAIL.NCIFCRF.GOV
Thu Jan 16 19:49:02 EST 1997


Our standard technique is as follows. We use this for PCR prior to SSCP
analysis or sequencing. Ask questions if you are not sure. We are always
willing to help.
Buz

DNA and RNA Extraction from Paraffin Sections

1/15/97 Greg Buzard, SAIC Frederick, NCI/FCRDC

DNA from paraffin sections of Ethanol, AMeX, Carnoy's or formalin-fixed
tissues may be amplified by PCR and utilized for determination of
mutations, etc. Other types of fixative, especially Bouin's and Zenker's,
are less well suited for DNA recovery. The type of tissue will determine
certain parameters.

1. Four to 10 micron-thick paraffin sections are cut with disposable blades
(per block) and floated onto sterile water in individual (per block)
plastic weighing boats. If the entire section is to be used, it can instead
be placed directly into a tube at this point. The sections that are to be
micro-dissected, or micro-selected by UV-protection (SURFED), are mounted
on sterile glass slides and heated gently to adhere them. PCR sterility
conditions are maintained, slides are never to be handled or exposed to air
in a PCR-evaluation room. Wear washed latex or vinyl gloves throughout
these procedures.

2. The first and last slides in the series are stained, usually H and E, to
be used as micro-selection guides (templates for tumor retrieval).

3. The desired areas of the section are individually scraped with a clean
razor or scalpel blade and put into a 0.5 ml tube (which may be
siliconized) containing 500 =B5l of n-octane (Octane appears to be better
than xylene). The scrapings may need to be washed from the blade into the
tube using the octane. Don't wash the area that may have been touched by
your glove (you are wearing your gloves, aren't you?). Alternatively,
pipette 0.5-1 =B5l of mineral oil onto the area to be scraped, use the same
tip to scrape and then collect the oil-suspended scrapings.

It is critical in this kind of work to have normal tissue controls
extracted simultaneously. A separate tube in which clean paraffin, or no
section, is added should be octane- and protease-treated just as are the
real samples. This no-template extraction-protocol-control extract should
be tested any time a new set of primers is used on DNAs extracted in this
series. Contamination during extraction of the templates is the most common
cause of "seeing the same mutation everywhere you look". Paraffin from the
edge of a tissue section is not a no-template control, since the microtome
blade drags DNA from the tissue across it. This is also the most common
source of mutations reported in "normal" surrounding tissues.

4. Vortex vigorously for 5-20 min (multiple tubes can be done in a 50 ml
conical centrifuge tube with a cap or in special foam rubber tube holders
found on some vortexers. Spin for 1-5 min at full speed, then carefully
pipette off the octane. Use filter-blocked tips for manipulations. Repeat
for larger amounts of paraffin.

5. Add 500 =B5l of 100% Acetone (not EtOH, which is not miscible with octane=
)
to the tube.=20

6. Vortex for 1-10 min, spin for 1-5 min, then pipette off Acetone.

7. Air-dry completely. Tubes and caps must be covered in parafilm with a
needle hole if speed-vac-ing. An air flow hood should be used if air drying
(sterility, sterility).

8. Add 100-400 =B5l (depending on how much tissue is being digested) of
digestion buffer (50 mM Tris pH 8.5, 1 mM EDTA, 0.5% Tween 20) and 2 =B5l of
Proteinase K stock solution (10 mg/ml). Leave out the Tween 20 if SDS is
going to be used, because it will counteract with the SDS. Incubate at 48
C for 1 hr to 5 days, depending on amount of tissue, type, fixative, etc.
For very difficult tissues, you may add a second aliquot of Proteinase K at
the end of one hour. Final concentration of proteinase K can be O.1-10=
 mg/ml.

Note that degraded RNA and DNA come off very rapidly, depending on the
tissue, the units of Proteinase K added, temperature and fixation. This
material is not what you want, and may be inhibitory to the PCR reaction.
It is the slower released higher molecular weight DNA that you want. Taking
a few test sections and sample the digest at 10 min, 30 min, 60 min, 4, 8,
24 and 48 hrs. This will allow you to monitor the beginning of HMW DNA
release. The material released prior to this should be discarded, fresh
reagent added, and the digestion continued to completion. This time period
is good only for this set of samples, and there may be variation within the
sets if tissues were handled differently (e.g. tissues were in formalin
different lengths of time before paraffin processing).

9. Spin for 1 min to collect condensate.

10. Heat at 95 C for 5-10 min (depending on protease concentrations used)
to inactivate the protease. Too hot or too long destroys the DNA, but you
must kill the protease before adding the Taq enzyme!=20

11. Buffers with SDS should be treated with Tween 20, or phenol/chloroform
extracted and EtOH precipitated. Spin at full speed for one minute to
pellet undigested material. Transfer DNA/RNA-containing supernate to a
clean tube. Store at -20 C, aliquot as needed to prevent the need for
freeze/thawing repeatedly. This DNA is good for at least one year when
stored at 4 C.

12. Test different volumes of samples/100 =B5l Rx vol., i.e., 5, 1, 0, 0.01
=B5l/100 =B5l reaction. Failure to make a PCR product is often because too m=
uch
of the supernate was added (some components of the digested extract are
inhibitory to the PCR reaction).

13. When performing the PCR reaction, take samples out at 20, 30, 40, 45,
50 and 55 cycles to determine how many cycles are optimal with a given
primer set and DNA template. Going to plateau is useful when dealing with
highly variable template qualities.




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