FFPE DNA extraction protocol
Contents
1. as they may still pop open from the thermal mixer and allow it cool at room temperature for 10 minutes Spin down the tube to remove any solution from the top of the tube 10 11 12 13 14 15 16 17 18 Appendix E FFPE DNA Extraction Protocol for OncoScan Assay 123 Add 20 uL of Proteinase K to the tube Vortex the tube at max speed for 10 seconds then spin down briefly Place tube on the thermal mixer that is set at 56 C Incubate the tube for at least 3 5 hours with a 15 second mix at 1400 rpm every minute After 3 5 hours verify that all tissue has lysed A If tissue remains incubate the samples overnight If tissue still remains add an additional 20 uL of Proteinase K and continue incubation for a minimum of 1 hour Spin down the tube and place them onto the thermal mixer that is set at 90 C Incubate the tube for 1 hour with a 15 second mix at 1400 rpm every 1 minute NOTE Thermomixer settings Ensure the thermomixer is programmed to shake the samples for 15 sec after each minute at 1400 RPM After 1 hour remove the tube from the thermal mixer and allow to cool at room temperature for 10 minutes Spin down the tube to remove any solution from the top of the tube Add 2 uL of RNase A to each tube Vortex the tube at max speed for 10 seconds then spin down briefly Allow to incubate for 2 minutes DNA Purification 1 10 11 12 13 14 Remove a QIAamp MinElute c2. 166 Block for 24 x 1 5 mL Tubes for Thermomixer R 22670522 Qty Required 2 VWR International 21516 176 Pipet Lite LTS Pipette Single Channel 0 5 10 uL Rainin L 10 Pipet Lite LTS Pipette Single Channel 20 200 uL Rainin L 200 Pipet Lite LTS Pipette Single Channel 100 1000 uL Rainin L 1000 Pipet Lite LTS Pipette 12 Channel 2 20 uL Rainin L12 20 Appendix E FFPE DNA Extraction Protocol for OncoScan Assay 121 Consumables Required Table E 2 Consumables Required Equipment Vendor P N HP 35 High Profile blades with PTFE coating Fischer Scientific 3153735 Eppendorf tubes 1 5 mL microcentrifuge tubes VWR International 022363204 Non Stick RNase free Microfuge Tubes 1 5 mL Life Technologies AM12450 Greenpak LTS 20 uL Filter Tip 960 Tips Rainin GP L10F Greenpak LTS 200 uL Filter Tip 960 Tips Rainin GP L200F Greenpak LTS 1000 uL Filter Tip 768 Tips Rainin GP L200F VWR PCR Plate 96 Well Flat Plate VWR 82006 636 Reagents Required Table E 3 Reagents Required Equipment Vendor P N QlAamp DNA FFPE Tissue Kit QIAGEN 56404 a QlAamp MinElute Columns 1020901 Collection Tubes 2 mL 1016810 Buffer ATL 1014758 Proteinase K 19133 Buffer AL 1014604 Buffer AW1 1014790 Buffer AW2 1014592 Buffer ATE 1049476 Xylene Sigma Aldrich 534056 500ML Ethanol 96 100 Sigma Aldrich 459836 RNase A QIAGEN 1007885 Optional Pre
3. FFPE DNA Extraction Protocol for OncoScan Assay f NOTE Affymetrix strongly recommends using the QlAamp DNA FFPE Tissue Kit protocol for purifying DNA from FFPE Blocks that will be used in OncoScan Assay For improved DNA yields we also recommend a modification to the QlAamp DNA FFPE Tissue Kit protocol The modified procedure adds a heating step at 98 C for 15 minutes to improve the tissue digestion process to release DNA from tissue sections Please refer to the QIAamp DNA FFPE Tissue Kit protocol for more information on the QIAGEN web page given below http www qiagen com Products Catalog Sample Technologies DNA Sample Technologies Genomic DNA QIAamp DNA FFPE Tissue Kit resources Equipment and Reagents Required but not Provided with OncoScan FFPE Assay Kit When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier Equipment Required Table E 1 Equipment Required Equipment Vendor P N Microtome HM340E Electronic Rotary Microtome Package with VWR International 89219 568 Universal Cassette Clamp and Disposable Blade Carrier E ThermoFisher Cat 905190A Vortex Genie 2 G560 VWR International 58815 234 Microcentrifuge 5424 EPPE022620401 VWR International 80094 126 Thermomixer R 22670107 Qty Required 2 VWR International 21516
4. ed 14000 rpm for 5 minutes Without disturbing the pellet remove the Xylene NOTE Be sure to place waste Xylene in the appropriate container Add 1 mL of Ethanol to the tube Vortex the tube at max speed for 10 seconds Spin down the tube at full speed 14000 rpm for 5 minutes Without disturbing the pellet remove the Ethanol by using a P1000 pipette NOTE Be sure to place waste Ethanol in the appropriate container Repeat Step 7 through Step 10 once more Spin down the tube at full speed 14000 rpm for 3 minutes Use a p20 or P200 pipette to completely remove any residual Ethanol without disturbing the pellet Allow any remaining Ethanol to evaporate by letting the tube air dry for 10 minutes at room temperature Add 180 uL of ATL Buffer to the tube after ensuring the residual ethanol has completely evaporated Vortex at full speed for 10 seconds Spin down the tube briefly and then place it onto the thermal mixer that is set at 98 C Incubate the tube for 15 minutes with a 15 second mix at 1400 rpm every 1 minute NOTE Thermomixer settings Ensure the thermomixer is programmed to shake the samples for 15 sec after each minute at 1400 RPM After 15 minutes stop the thermomixer program and turn off the thermomixer Let the tube cool down for 5 minutes in the thermomixer before removing them This is to ensure the vials do not pop open due to high heat and pressure Remove the tube carefully and slowly
5. olumn from the refrigerator and allow to warm to room temperature for 15 minutes Remove the ATL tube and equilibrate at room temperature Add 200 uL of Buffer AL to the sample tube vortex at max speed for 10 seconds then spin down briefly A If processing multiple samples ensure that after adding Buffer AL Ethanol is added as quickly as possible B Precipitate may form at this step which does not affect the DNA yield Add 200 uL of Ethanol to each tube vortex at max speed for 10 seconds then spin down briefly Label the QIAamp MinElute column in a 2 mL collection tube appropriately 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 A Check to see that all the lysate has moved through the column If lysate is still in the column centrifuge again at a higher speed for 1 minute Place the column into new collection tube and discard eluate Open the column and add 500 uL of Buffer AW1 Load the column onto the centrifuge and spin at 8000 rpm 6000g for 1 minute A Check to see that the entire Buffer AW1 has moved through the column If Buffer AW1 is still in the column centrifuge again at a higher speed for 1 minute Place the column into new collection tube and discard eluate Open the column and add 500 uL of Buffer AW2 Load the column onto the centrifuge and spin at 8000 rpm 6000g for 1 minu
6. requisite The yield of tissue from FFPE is highly dependent on the type of tissue and method used for the initial fixation For optimal DNA yield samples submitted for extraction should have a tissue size of 400 700 square mm Preparation of Buffers Preparing Buffer ATL Check whether precipitate has formed in Buffer ATL If necessary dissolve by heating to 70 C with gentle agitation Preparing Buffer AL a Check whether precipitate has formed in Buffer AL If necessary dissolve by heating to 70 C with gentle agitation Appendix E FFPE DNA Extraction Protocol for OncoScan Assay 122 Preparing Buffer AW1 Add 25 mL ethanol 96 100 to the bottle containing 19 mL Buffer AW1 concentrate Can be stored in room temperature up to year Preparing Buffer AW2 Add 30 mL ethanol 96 100 to the bottle containing 13 mL Buffer AW2 concentrate Can be stored in room temperature up to 1 year Deparaffinization 1 Au PWN 10 11 12 13 14 Tissue Lysis 1 2 3 4 From an FFPE block prepare 10 micron slices Place 5 slices ina 1 5 mL Eppendorf Safe Lock Tube f NOTE The Eppendorf Tubes must have the centrifugation stability of up to 30 000 x g to prevent tube breakage Please refer to the consumables list Turn on two thermal mixers Set one to 56 C and the other to 98 C Add 1 mL of Xylene to the tube Vortex the tube at max speed for 10 seconds Spin down the tube at full spe
7. say Kit Catalog Number P7589 LifeTechnologies a Qubit dsDNA HS Assay Kit Catalog Number Q32851 LifeTechnologies Record the results and the volume information in a spreadsheet
8. te A Check to see that the entire Buffer AW2 has moved through the column If Buffer AW2 is still in the column centrifuge again at a higher speed for 1 minute Place the column into new collection tube and discard eluate Load the column onto the centrifuge and spin at 14000 rpm 20000g for 3 minutes to dry the membrane completely DNA Elution wv PWM Appendix E FFPE DNA Extraction Protocol for OncoScan Assay 124 Label a clean Nuclease free 1 5 mL tube for DNA elution Place the column into the labeled 1 5 mL tube prepared for elution Add 50 uL of Buffer ATE to the center of the column membrane Close the lid and incubate the column at room temperature for 5 minutes Load columns onto the centrifuge and spin at 14000 rpm 20000g for 1 minute to elute the DNA A Note Caps for 1 5 mL tube will not be able to close at this step due to the columns When loading onto the centrifuge rotate caps to the right to keep them from breaking during the spin B When eluting multiple samples alternate positions in centrifuge to make room for caps Quantitation of Eluted FFPE DNA 1 Perform either of the two Affymetrix recommended and tested dsDNA quantitation protocols that are included in the user manual to measure the concentration of the eluted FFPE DNA Affymetrix strongly recommends using these protocols that have been tested at Affymetrix using the following kits for DNA quantitation Quant iT PicoGreen dsDNA As
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