A simple method for DNA extraction from formalin-fixed, paraffin embedded tissue blocks
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Tissue preparation
- We used FFPE tissue blocks of Non-small cell lung cancer obtained from Pathology department of Kerman Medical University which were fixed from 2004 to 2006.
- Before outset of our examination, all plastic microtube (1.5ml and 0.5 ml) and pipette tips were autoclaved as well as sterile distilled water used.
- After delivering tissues from Pathology department, tissues were carved with a surgical blade into 1mm pieces and between 25 mg to 75 mg of the tissue was transferred into a microtube. The tissues were deparaffinized as follows.
Deparaffinization method
- Tissue sections were deparaffinized with 500 microliter 100% xylene under a fume hood. Then contents were vortexed for 1 minutes and left in a 65 ºC water bath for 15 minutes.
- The xylene solvent was decanted immediately and the procedure repeated two more times.
Xylene removal
To remove the residual xylene, the samples were washed five times with Ethanol as follows.
- 1ml of absolute ethanol was added and mixed by vortex for ten seconds and removed after 10 minutes.
- 1ml of absolute ethanol was added and mixed by vortex for ten seconds and removed after 30 minutes.
- 1ml of 90% ethanol was added and mixed by vortex for ten seconds and after 20 minutes, tube was centrifuged at 8000 RPM and then removed ethanol out.
- 1ml of 70% ethanol was added and mixed by vortex for ten seconds and after 20 minutes, tube was centrifuged at 8000 RPM and then removed ethanol out.
- 1ml of 50% ethanol was added and mixed by vortex for ten seconds and after 20 minutes, tube was centrifuged at 8000 RPM and then removed ethanol out.
Tissue lysis
- After the tissues were dried, 500 microliter lysis solution was added to each microtubes (40 mM Tris, 1mM EDTA, 0.5% Tween-20, 0.5 µg/µl proteinase k, PH,8) the proteinase k was added after pH adjustment and the microtubes were left in a 60 ºC water bath and were inverted every 30 minutes until the tissue was completely lysed (temperatures over 60 will inactivate the enzyme rapidly.
- Then resulting cell lysate was heated at 95 ºC for 8 minutes to inactivate the proteinase k.
Phenol-cholorophorm method
For the DNA extraction from cell lysate, phenol-chloroform was used as following.
- An equal volume of Tris-saturated phenol (pH,8) was added the tubes were left on a rotating wheel for 10 minutes then centrifuged at 12000 for 2 minutes.
- Then 300 microliters of the upper phase was transferred to a new microtube and an equal volume of Phenol-Cholorophorm mixture (1:1) the tube contents were mixed on the rotating wheel for 10 minutes and were centrifuged at 12000g for 2 minutes.
- The upper phase transferred to new microtube and 2.5 volume of cold absolute ethanol was added and For DNA precipitation, microtube was left overnight at -20 ºC.
- Then tubes were centrifuged for 30 minutes at 4 and ethanol was decanted.
- The DNA pellet was washed gently two times with cold 70% ethanol and then the pellet was dried completely at room tempreture.
- The pellet was dissolved in 30 to 70 microliters of steril destilled water, depending on the size.
- To complete the solubilization the microtubes were put in 40 ºC water bath for one hour.
- To check the extracted DNA, 5 microliters of the solution was run on a 1% agarose gel. The extraction DNA is suitable for PCR.
References
- Cawkwell L, Quirke. Direct multiplex amplification of DNA from formalin fixed, paraffin wax embedded tissue section, J Clin Pathol: Mol Pathol 53, 51-52, 2000.
- Chan P K S, Chan D P C, To K-F, et al. Evaluation of extraction methods from paraffin wax embedded tissues for PCR amplification of human and viral DNA. Clin Pathol 54, 401-403, 2001.
- Jung K, Ha Y, Kim S-H, et al. Development of Polymerase Chain Reaction and comparison with In situ Hybridization for the detection of Haemophilus parasuis in formalin-fixed, Paraffin embedded tissues, J.vet. Med. Sci. 66(7), 841-845, 2004.
- Keohavong P, Gao W-M, Zheng K-C, et al. Detection of K-ras and p53 mutations in sputum samples of lung cancer patients using laser capture microdissection microscope and mutation analysis, Analytical Biochemistry 324, 92-99, 2004.
- Shi S-R, Cote R J, Wu L, et al. DNA extraction from archival formalin-fixed, Paraffin-embedded tissue sections based on the antigen retrieval principle: Heating under the influence of pH, The journal of Histochemistry & Cytochemistry 50, 1005-1011, 2002.