Funded by European Union Horizon 2020 Grant No 825771
Translational studies of HEAD and neck cancer in
South America and Europe
SOP for DNA extraction from
Formalin-Fixed Paraffin-Embedded (FFPE) tissue
Version 1.0 – May 2020
Deliverable No.
D3.1 Part 3
Deliverable title
SOPs for FFPE tissue accession sectioning, tissue array processing,
and storage
Responsible partner
University El Bosque (UnBosque)
Contributors
Hospital Universitario Fundación Santa Fe de Bogotá (FSFB),
Catalan Institute of Oncology (ICO), University of Turin (UNITO),
Hospital Santa Rita de Cassia (AFECC), University of Bristol (UBRIS)
Contents 1. Introduction 3 2. Objective 3 3. Test specifications 3 4. Required equipment 3 4.1 Reagents/materials 3 5. Processing protocol 4 5.1 Reagent Preparation 4
5.2 Preparation of tissue sections 4
5.3 DNA Extraction 6
5.4 DNA purification 6
6. Quality controls 7
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1.
Introduction
The purpose of this SOP is to describe the instructions for DNA extraction from FFPE tissue.
2.
Objective
Define the procedure and establish the basic quality guidelines regarding DNA extraction from FFPE tissue.
3.
Test specifications
• Test type: Molecular Biology Study
• Name of study: DNA extraction from FFPE tissue (KIT: PINPOINT SLIDE DNA ISOLATION SYSTEM ZYMO RESEARCH CATALOG No. D3001)
• Other kits to use:
• Qiagen: QIAamp DNA FFPE Tissue Kit - Cat No./ID: 56404
• Epibio: QuickExtract™ FFPE DNA Extraction Kit - Cat. Nos. QEF8180 and QEF81050
• Type of sample: Formalin-Fixed Paraffin-Embedded (FFPE) tissue
4.
Required equipment
• Freezer at ~ -20 ° C
• Laminar flow cabinet
• Microcentrifuge • Vortex • Wet bath • Dry bath 4.1
Reagents/materials
• CONSUMABLES o Pipettes o Pipette tips o Sterile blades o Sterile 1.5ml tubes• REAGENTS AND SUPPLIES CONTAINED IN THE KIT o 1 Pinpoint solution tube
o 1 Proteinase K (lyophilized) set + Storage Buffer o 2.5 ml PP Extraction Buffer
o 6 ml Pinpoint Binding Buffer
o 2.4 ml PP Wash Buffer (concentrated) o 50 Zymo-spin I columns
o 50 Collection tubes
• REAGENTS AND SUPPLIES NOT CONTAINED IN THE KIT o Ethanol (50%, 70%, 95-100%)
o Xylol (95-100%)
o Water degree Molecular Biology
5.
Processing protocol
5.1
Reagent Preparation
• Preparation of Proteinase K: Add 260ul of the "Proteinase K Storage Buffer" to the lyophilized Proteinase K tube. Dissolve completely and store at -20 °.
• Preparation of the “PP Wash Buffer”: Add 14ml of 95-100% Ethanol to the concentrated “PP Wash Buffer”.
5.2
Preparation of tissue sections
To avoid the cross contamination, consider the following recommendations given by the HPV-AHEAD consortium. The following protocol describes the generation of several sections for laboratory assays and histological analyses, as schematically shown in Fig 1 (Mena M, et al. (2017). PLOS ONE 12(10))
• According to the size of tissue available, make between 10 and 31 sections
• Use the S6 to S8 sections for detection of and genotyping HPV DNA (see figure 1).
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✔ Phase 1: Preparation of all the tumour paraffin blocks
BLANK
TUMOUR
✔ Phase 2: Sectioning protocol
Process blank block after
every 10 tumors blocks
- Step 6: Wear new disposable gloves and insert a
new microtome blade
- Step 7: Mount blank paraffin block
- Step 8: Generate three consecutive sections of 10um tick.
- Step 9: Collect in a new sterile 1.5mL Eppendorf
tube using clean Millipore tweezers (extensively washed with DNA cleaner, followed by 70% ethanol).
- Step 10: Change the microtome blade.
- Step 6: Wear new disposable gloves and insert a new
microtome blade
- Step 7: Mount tumour paraffin block
- Step 8: Generate two consecutive sections of 5um tick and mount them and coated slides for histology and p16 staining (S1 and S2).
- Step 9: Generate three consecutive sections of 10um
thick for RNA extraction (S3 to S5)
- Step 10: Clean carefully the Millipore tweezers with DNA cleaner followed by 70% ethanol.
-Step 11: Collect with clean Millipore tweezers (extensively washed with DNA cleaner, followed by 70% ethanol) the sections in a new sterile 1.5mL Eppendorf tubes.
- Step 12: Generate three consecutive sections of
10um thick for DNA extraction (S6 to S8)
-Step 13: Collect with clean Millipore tweezers
(extensively washed with DNA cleaner, followed by 70% ethanol) the sections in a new sterile 1.5mL Eppendorf tubes.
-Step 14: Generate two consecutive sections of 5um
thick and mount coated slides for p16 staining and histology (S9 and S10). (In case the specimen size is
limited, please stop at step 14, otherwise progress to step 15).
-Step 15: Warning, it is likely that after 10 sections the
block needs to be cooled down on -20°C plate.
- Step 16: Generate twenty consecutive sections of
5um thick mount them on coated slides for immunohistochemical analyses (S11-30).
-Step 17: Generate one final section of 5um thick for
histology (S31).
Return to step 5 and process a new specimen.
-Step 1: Prepare the cover slides with the right ID numbers
-Step 2: Wear disposable gloves, remove the used blade, and clean the microtome (especially the surface where the slides are collected and the holder - forceps), and the bench with DNA cleaner and 70% ethanol using disposable tissue papers. Discard cleaning tissue papers and gloves.
-Step 3: Wear new disposable gloves and insert a new microtome blade. -Step 4: Remove the rough surface and flatten all the blocks.
-Step 5: Remove the used blade and clean the microtome and the bench with DNA cleaner and 70% ethanol using disposable tissue papers.
✔ Also consider the following indications for DNA extraction:
• Is important to wash the sectioning platform and microtome blade extensively with DNA cleaner and 70% ethanol and wear a fresh clean lab coat and face mask between each different sample.
• Mount 10um thick sections on unloaded glass sheets and let dry for 30 minutes at 60° C.
• To dewax the tissues, place the sheets in xylol at room temperature for 30 minutes, then change the xylol and incubate for another 30 minutes.
• Rehydrate the cuts by passing the sheets through 100% ethanol, 70%, 50% and sterile water, for 2 minutes each.
• Let the cuts air dry.
• Mark on the back of the sheet (with indelible marker) the area of tissue from which DNA is to be extracted, as indicated by the pathologist on the hematoxylin-Eosin staining of the tissue.
• Apply the Pinpoint solution over the selected area: Use a pipette tip for the application, leaving a very thin layer.
• Allow the Pinpoint to dry at room temperature: at this point the tissue cells are included within the Pinpoint film.
• Remove the tissue from the sheet (microdissection): use a clean blade for each case and transfer the tissue to a properly labeled 1.5ml tube. Take approximately 20mm2 (between 4 and 8 cuts).
• Short centrifuge to send the tissue fragments to the bottom of the tube. 5.3
DNA Extraction
• Add 50ul of “Extraction Buffer” and 5ul of Proteinase K to the tube with the tissue, mix by vortex and make a short spin.
NOTE: for multiple samples, the “Extraction Buffer” and Proteinase K can be mixed beforehand, and 55ul of the mixture added to the sample.
• Incubate the tubes at 55 ° C for at least 4 hours in a water bath.
• Heat the tubes for 10 minutes at 98 ° C and then immediately put on ice (This can be done in a thermal cycler, for this purpose, the contents of the 1.5ml tubes must be previously transferred to 0.2ml tubes). Ensure that the temperature is above 95 ° C, because incomplete inactivation of Proteinase K can cause problems in the PCR reaction.
• Make strong vortex for 10-15 seconds. Short centrifuged.
NOTE: According to the indications of the KIT, in most cases at this point the DNA is ready for PCR analysis. According to the experience of the area of Molecular Biology, the subsequent DNA Purification should always be performed.
5.4
DNA purification
• Add 100ul of “Pinpoint Binding Buffer” to the sample previously treated with Proteinase K, and mix.
• Transfer the mixture to the “Zymo-Spin I Column”, properly labeled, assembled on a 2ml collection tube.
• Centrifuge at maximum speed for 10 seconds.
• Add 150ml of “PP Wash Buffer”, previously prepared, to the column and centrifuge at maximum speed for 10 seconds.
• Add 150ml of “PP Wash Buffer” again, and centrifuge at maximum speed for 1 minute.
• Transfer the column to a sterile 1.5ml tube, properly labeled.
• Add 20ul of water, TE Buffer or Elution Buffer directly on the column membrane, without touching it, wait for 1 minute, and centrifuge at maximum speed for 10 seconds to recover the DNA.
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Note: For the DNA that will eventually be taken for further analysis, it is recommended to elute in distilled water free of nucleases. TE buffer with very low concentration of ETDA and nuclease free can be used but with caution (10mM Tris-HCI, pH8.0, 0.01mM EDTA).
CAUTION: If the TE buffer has EDTA concentrations of 0.1-1mM, it captures magnesium-free ions, which reduces the activity of DNA polymerase, resulting in sequence reaction failures.
• Quantify the DNA obtained and then adjust its concentration according to the conditions of the analysis to be performed.
6.
Quality controls
Negative extraction control: to ensure that the reagents are not contaminated.
7.
References
• Buton, M.P., Schneider, R., Brown, N., Escamilla-Ponce y M.L. Gulley. 1998. Comparison of histologic stains for use in PCR analysis of microdissected, paraffin-embedded tissues. Bio Techniques 24:86-92.
• Greer, C.E., J.K. Lund and M. Manos. 1991. PCR amplification from paraffin- embedded tissues: recommendations on fixatives for long-term storage and prospective studies. PCR Methods Appl. 1:46-50.
• Mena M, Lloveras B, Tous S, Bogers J, Maffini F, Gangane N, et al. (2017) Development and validation of a protocol for optimizing the use of paraffin blocks in molecular epidemiological studies: The example from the HPV-AHEAD study. PLoS ONE 12(10): e0184520. https://doi.org/10.1371/journal.pone.0184520