How to Resolve DNA Extraction Failures in FFPE Tissue Processing?

Starting material was too much so modification to the protocol were applied

Add 1 ml xylene to the sample. Close the lid and vortex vigorously for 10 s.

Centrifuge at full speed for 2 min at room temperature (15–25°C).

Remove the supernatant by pipetting. Do not remove any of the pellet.

Add 1 ml ethanol (96–100%) to the pellet, and mix by vortexing. The ethanol extracts residual xylene from the sample. (After adding the ethanol, the top of the tissue had solidified because there was too much wax. The ethanol has been removed and the xylene step has been repeated other 2 times to remove as much wax as possible).

Add 1 ml ethanol (96–100%) to the pellet, and mix by vortexing. The ethanol extracts residual xylene from the sample.

Centrifuge at full speed for 2 min at room temperature.

Remove the supernatant by pipetting. Do not remove any of the pellet. Carefully remove any residual ethanol using a fine pipet tip.

Open the tube and incubate at room temperature or up to 37°C. Incubate for 10 min or until all residual ethanol has evaporated.

Resuspend the pellet in 180 µl Buffer ATL. Add 20 µl proteinase K, and mix by vortexing. (the pellet has been resuspended with 360 µl Buffer ATL and 40 µl proteinase K as there was too much material)

Incubate at 56°C for 1 h.

Incubate at 90°C for 1 h.

Prepare a mix of 200 µl buffer AL and 200 µl ethanol 100% per sample (in this case has been prepare 1400 µl of mix for )

Briefly centrifuge the 1.5 ml tube with the sample to remove drops from the inside of the lid (each sample has been spinned down and split into two 1.5 ml tubes to avoid clogging of the column in the next steps).

Add 400 µl Buffer AL/Ethanol mix to the sample, and mix thoroughly by vortexing.

Briefly centrifuge the 1.5 ml tube to remove drops from the inside of the lid.

Carefully transfer the entire lysate to the QIAamp MinElute column (in a 2 ml collection tube) without wetting the rim, close the lid, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp MinElute column in a clean 2 ml collection tube, and discard the collection tube containing the flow-through. If the lysate has not completely passed through the membrane after centrifugation, centrifuge again at a higher speed until the QIAamp MinElute column is empty.

Carefully open the QIAamp MinElute column and add 500 µl Buffer AW1 without wetting the rim. Close the lid and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp MinElute column in a clean 2 ml collection tube, and discard the collection tube containing the flow-through.

Carefully open the QIAamp MinElute column and add 500 µl Buffer AW2 without wetting the rim. Close the lid and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp MinElute column in a clean 2 ml collection tube, and discard the collection tube containing the flow-through.

Centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min to dry the membrane completely.

Place the QIAamp MinElute column in a clean 1.5 ml microcentrifuge tube, and discard the collection tube containing the flow-through.

Carefully open the lid of the QIAamp MinElute column and apply 60 µl RNeasy free water to the center of the membrane. (because the samples were split into two, each sample has been eluted with 30 µl of RNasy free water to concentrate it)

Close the lid and incubate at room temperature for 5 min.

Centrifuge at full speed (20,000 x g; 14,000 rpm) for 1 min.

The six samples (two duplicates) has been quantified with the Nanodrop, the duplicates has been then pooled in one vial (final volume of around 50 µl ) and quantified again with Nanodrop.

Kayvan Masoudi

Hello,

I would like to express my gratitude for your explanations and guidance.

Following the protocol you suggested, the deparaffinization step was carried out correctly, resulting in dry tissue without paraffin residue. Subsequently, I ground the tissue into powder using a mortar and pestle. Following your recommendation, I transferred a smaller amount of tissue to the microtube for lysis, added the lysis buffer and proteinase K, and incubated it at 60 degrees Celsius for 3 hours and then at 70 degrees Celsius for 1 hour. However, despite these steps, the tissue is not lysing, and it remains suspended in the lysis buffer.

Kayvan Masoudi

This is the extraction kit that I use

FAVORGEN “FavorPrep Tissue Genomic DNA Extraction Mini Kit”

Ilaria Mulas

Hello Kayvan Masoudi :)

I'm sorry that you are still having issues with your extraction.

Do you use a piece of tissue or microtomy sections?

With my protocol, I cut microtomy sections of 5 microns, in order for the tissue to be thin and I don't need to use mortar and pestel. I don't use too much tissue (max 6 sections) otherwise it'll clog the columns.

If you are using too much tissue maybe it's the reason why the lysis is not complete.

I use the Qiagen DNA FFPE extraction kit

QIAamp DNA FFPE Tissue Kit | FFPE DNA Extraction| QIAGEN

Cat. No. / ID: 56404

Let me know if you need more advice and if you get good results.

Best wishes

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