- 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.
- 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.
To remove the residual xylene, the samples were washed five times with Ethanol as follows.
The microtubes were then left in a 40 ºC oven to dry the tissues.
- 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.
- 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.
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.
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- 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.