truXTRAC FFPE DNA 8 microTUBE Strip Kit for chemagen
Contents
1. Option A Shear DNA during extraction to a size suitable for next generation sequencing library construction Fragment size can be tuned between 200 and 400 bp Option B Extract 2kb DNA fragments This protocol is recommended for most analytical applications including PCR Note that actual DNA fragment size will depend of the quality of the starting material Option C Extract large genomic DNA without any additional fragmentation Actual DNA fragment size will depend on the quality of the starting material For high quality FFPE tissue blocks we typically see an average fragment size of gt 8 kb Please refer to Appendix A for examples of final DNA fragment size distribution OPTION A EXTRACT AND FRAGMENT DNA FoR NGS Load FFPE sample section or core into microTUBE Remove paraffin and rehydrate tissue with AFA KS Digest with Proteinase K Le Reverse crosslinks A Fragment DNA to desired size with AFA Le Purify DNA Part Number 010295 RevA 5 Page July 2015 Patents Granted and Pending OPTION B EXTRACT LARGE DNA FRAGMENTS gt 2 KB WITH IMPROVED YIELD Load FFPE sample section or core into microTUBE v Remove paraffin and rehydrate tissue with AFA KS Digest with Proteinase K Se Reverse crosslinks A Release of DNA with AFA KS Purify DNA Actual DNA fragment size will depend of the quality of the starting tissue block OPTION C EXTRACT GENOMIC DNA Loa2. 8 Repeat steps 3 7 with an initial set point of 90 C to obtain T set T set C 180 C Tth Part Number 010295 Rev A 19 Page July 2015 Patents Granted and Pending Additional Notes 1 Covered by US Patent 9 080 167 2 Other patents pending 3 Best Practices for determining the yield and purity of isolated DNA e To determine DNA yield with the highest level of accuracy a fluorometric assay such as Qubit Life Technologies should be used e In addition spectrophotometric analysis of DNA for A260 280 and A260 230 ratios will determine if protein or peptide salt contamination is present in the sample 4 Tissue Blocks were obtained from Theresa Kokkat PhD and Diane McGarvey Cooperative Human Tissue Network CHTN Eastern Division University of Pennsylvania USA 5 See following link http covarisinc com wp content uploads pn_010295 pdf for updates to this document 6 The treatment settings listed in this document are recommended guidelines Actual results may vary depending on the tissue type mass and previous handling of FFPE samples Part Number 010295 RevA 20 Page July 2015 Patents Granted and Pending
3. AMPLES OF DNA FRAGMENTS SIZE DISTRIBUTION Multiple 10 um sections were cut off the same FFPE kidney tissue block and DNA was extracted with the Covaris FFPE kit using Option A B or C Extracted DNA was purified and analyzed ona Bioanalyzer The size of the non fragmented genomic DNA Option C depends on how the tissue block was generated and stored as well as the age of the tissue block 200 300 and 400 bp Peaks E Option A Extract and fragment DNA for NGS After extraction DNA was sheared to a size suitable for NGS library construction In this example one sample was sheared to 200 bp one to os SEN 300 bp and one to 400 bp a 1 BS 700 100 as Sein EAR 17000 bp bel Sample 12 C Option B Extract large DNA 70 3 kb Peak 504 i TNI ft os E EE 3000 17000 bp 104 E T T T se 50 300 500 700 1000 fragments gt 2 kb After extraction AFA energy was used to increase DNA release from the tissue During this process the DNA was also sheared into fragments larger than 2 kb Final size will depend of the quality of the starting tissue 8 kb Peak f f G 1 SE ene E EE SESS Epa OS SS Kg r M Lem E E EE s 100 30 emm 700 wun 200 e 17000 te Option C Extract genomic DNA No additional AFA energy was applied after extraction DNA size will be the largest possible Final size will depend of the quality of the starting tissue
4. NA Size length loading Collection tool tissuePICK sectionPICK NA Maximum ber of 2x tissuePICK number o 200 mm tissue TRIMMED 2x sectionPICK 2 1 1 for a 5 um samples Per section Tube NOTE For optimal tissuePICK and sectionPICK performances tissue section should be mounted on uncoated slides The tissuePICK and sectionPICK should always be used in conjunction with a sectionWARMER Part Number 010295 Rev A 7 Page July 2015 Patents Granted and Pending 2 Tissue Fixation Requirements The yield and quality of DNA extracted from FFPE tissue blocks is highly dependent on tissue collection and paraffin embedding procedures For good yields of high quality DNA Use a maximum fixation time of 24 hours Use Formalin solution neutral buffered 4 Fix sample tissue sample as quickly as possible after collection Buffer 1 Check Tissue SDS Buffer A white precipitate may form during storage Incubate the bottle at 50 70 C before use to dissolve any precipitate Instruments NOTE For detailed instructions on how to prepare your particular instrument please refer to your instrument s User Manual 1 For E and LE Series Focused ultrasonicators fill the water bath set the chiller temperature as described in Table 1 and allow the system temperature to equilibrate and the water bath to degas For E210 or E220 load the plate definition E220 500444 Rack 12 Pla
5. PROTOCOL Covaris truXTRAC FFPE DNA 8 microTUBE Strip Kit for chemagen Technology 96 Adaptive Focused Acoustics AFA based DNA extraction and purification from Formalin Fixed Paraffin Embedded FFPE Tissue Contents INTENDED EE 2 INTRODUCTION Ee ee Eege E 2 REVISION HISTORY EE 3 KIT CONTENTS eneen RER EA EE O ONEA NOAOA reegt ebe Eer 3 eeler 3 SUPPLIED BY USERS eebe Geet eege eet eet dca gege Eed ee gege e eet 3 PROCEDURE WORKFLOW OVERVIEW soriire rair iee EER EA AREE R EEE E ARA E ER A 5 T PREPARATION oriana anr Ee Ea eN EE AAN E AAA ENSE AANEREN E AN AEO AAS AA TS AA AAA Aa ENEE a 7 ORT UE H BEE ergeidege gees E E IE OE EN E E EA enee ENEE E eege eeh 8 lee gf a EE 8 Prepare Heating Bleck et Seegen gedeien EEN 9 PerkinElmer chemagic Drepito g D 9 2 DNA EXTRACTION FROM FFPE TISSUE c sssccecssssececsesceecsesaceecseueeecsesueeecsesueeecsesueeecsesueeeceeaueeesseqaeeeeeee 10 APPENDIX A EXAMPLES OF DNA FRAGMENTS SIZE DISTRIBUTION ccseeececeesseeecesseeeeceeseeeecesseeeecesseeaeceseenaeens 16 APPENDIX B EXAMPLE OF PARAFFIN EMULSIFICATION WITH AFA ENERGY csecccceseeeecesseececeseeeeecesseaaeesseeaees 17 APPENDIX C TROUBLESHOOTING GUIDE 18 APPENDIX D COVARIS HEAT BLOCK NOMINAL DIMENSIONS 19 APPENDIX E DRY BLOCK HEATER CALIBRATION PROCEDURE 19 Part Number 010295 RevA 1 Page July 2015 Patents Granted and Pending INTENDED USE The truXTRAC FFPE DNA Kit is intended for use in m
6. Part Number 010295 RevA July 2015 Patents Granted and Pending 16 Page APPENDIX B EXAMPLE OF PARAFFIN EMULSIFICATION WITH AFA ENERGY Paraffin was emulsified in a microTUBE Screw Cap using a Covaris S220 Focused ultrasonicator Sample before left side and after right side processing Sample was a 10 um kidney tissue section Part Number 010295 RevA 17 Page July 2015 Patents Granted and Pending APPENDIX C TROUBLESHOOTING GUIDE Issue Cause Solution Comments Suggestions Low yield of Low tissue to wax Repeat the procedure using In your initial use of the DNA ratio in FFPE additional sections until truXTRAC FFPE kit use section desired yield is achieved FFPE blocks that have been well characterized for yield and quality Proteinase K stored above recommended temperature or expired Repeat the procedure using fresh Proteinase K Always store proteinase K solution at Room Temperature or 4 C DNA does not DNA in FFPE Design amplicons to be as small DNA isolated using Covaris perform well sample blocks is as possible lt 100 bp AFA technology is of the in severely cross highest possible quality downstream linked or Some FFPE sample blocks applications degraded may be too degraded or such as qPCR cross linked for some applications DNA Too much Trim any excess paraffin from Too much emulsified fragment size emulsified tissue blocks before proceeding paraffin abso
7. ble 3 Paraffin removal and tissue rehydration settings Duty Peak Incident Cyclesper Treatment Temperature Factor Power burst Time Instrument 10 175 Watts 200 300 sec 20 C 10 5 Intensity 200 300 sec 20 C 20 450 Watts Since PIP is distributed across multiple microTUBEs in an LE220 the power received by individual microTUBE stays within the 200 W limit 11 Protein digestion at T set IMPORTANT Use a Covaris Heat Block inside the dry block heater instead of a standard heat block for microfuge tubes Calibrate the dry block heater before use see Appendix E for instructions Failure to calibrate the dry block heater may lead to incomplete tissue digestion and or incomplete crosslink reversal Part Number 010295 RevA 12 Page July 2015 Patents Granted and Pending Insert a Covaris Heat Block into a dry block heater set at temperature T set Once T set has been reached load the rack containing the samples into the Covaris Heat Block An incubation time of 1 hour at T set is sufficient for sections 10 um or less in thickness 12 hour i e overnight incubation should be used for larger samples such as 25 um sections and cores If the digestion is incomplete after 12 hours add an additional 20 ul of Proteinase K solution mix and incubate for 1 more hour 12 Incubate the samples at T set for 1 hour to reverse formaldehyde crosslinks a Insert a Covaris Heat Block into a dry block heater se
8. ce 8 microTUBE Strip V2 1 5mm offset and check that the intensifier is in place For E220 evolution load the plate definition 500437 Rack E220e 8 microTUBE Strip V2 6mm offset and check that the intensifier is in place For LE Series Load the plate definition LE220_500485 Rack XT 12 Place 8 microTUBE Strip V2 1 5mm offset NOTE If you do not see the correct plate definition on your system please contact Covaris technical support at TechSupport covarisinc com Table 1 Focused ultrasonicator setup Instrument Water level Fill RUN scale Chiller temperature E Series amp L Series 6 18 C Part Number 010295 RevA 8 Page July 2015 Patents Granted and Pending Prepare Heating Block Dry block heaters should be preset at T set and T setz See Appendix D and E for instructions on how to calibrate and install the Covaris Heat Blocks PerkinElmer chemagic Prepito D Please refer to the chemagic Prepito D instrument user manual revision 2022 0020 Part Number 010295 RevA 9 Page July 2015 Patents Granted and Pending 2 DNA EXTRACTION FROM FFPE TISSUE 1 Using Table 2 below as a guide generate the Processing Buffer master mix by mixing Tissue Lysis Buffer and Proteinase K Table 2 Processing Buffer master mix Number of Tissue SDS Buffer Proteinase K volume samples volume 8 16 1408 ul D x 88 ul x 22 ul 2 Carefully peel off the blue tape from the 8 microTUBE 130 St
9. d FFPE sample section or core into microTUBE A Remove paraffin and rehydrate tissue with AFA b g Digest with Proteinase K P Reverse crosslinks A Purify DNA Actual DNA fragment size will depend of the quality of the starting tissue block Part Number 010295 RevA 6 Page July 2015 Patents Granted and Pending 1 PREPARATION FFPE Tissue Sample 1 Sample Input requirements The truXTRAC process is highly efficient at removing paraffin even from relatively thick FFPE sections while simultaneously rehydrating the tissue Use of thicker sections is often desirable both for increased yield and since DNA or RNA in the exposed surfaces of a section tends to degrade quickly We recommend using sections between 15 and 25 um thick or cores of 1 2 mm IMPORTANT Excess paraffin will adversely affect the yield and quality of DNA and RNA extracted from FFPE We strongly advise trimming off any excess of paraffin before sectioning a FFPE tissue block or after the section has been cut from the FFPE block A ratio of 80 tissue to 20 paraffin or higher is ideal The total mass of FFPE sample processed per extraction should be between 2 to 5 mg Lower amounts may result in insufficient yield FFPE Sections FFPE Sections Mounted on slide scrolls or curls Size thickness lt 1 2mm g 4 to 10 um 7 to 10 um 7to15um 16 to 25 um or diameter diameter lt 10 mm if longer cut in half before
10. ity E210 Duty Factor Cycle per Burst Temperature LE220 Focused ultrasonicator Targeted fragment size 200 bp 300 bp 400 bp Treatment Time 450 sec 225 sec 120 sec PIP 450 W 450 W 450 W Duty Factor 20 20 20 Cycle per Burst 200 200 200 Temperature 20 C 20 C 20 C Part Number 010295 RevA 14 Page July 2015 Patents Granted and Pending 3 DNA PURIFICATION WITH CHEMAGEN TECHNOLOGY This step requires the Prepito truXTRAC DNA FFPE Kit CMG 2037 and associated script 4 Place the rack with the samples on a Covaris Heat Block set at T set to prevent paraffin from solidifying If amounts of emulsified paraffin are small it may be possible to keep the samples at room temperature Transfer the FFPE lysates to the first row of a 96 well Deep Well Plate DWP included in the Prepito truXTRAC DNA FFPE Kit Sample should be retrieved by pipetting through the septum For easy pipetting of sample in a single step we recommend using pipette tips with ribs e g Hamilton 250 ul Ribbed Tip or Agilent Bravo 250 ul Tip Otherwise two pipetting steps with 50 ul tips may be necessary Optional The sample can be treated with RNase A to remove RNA before DNA purification Add 5ul of RNase A solution to the FFPE lysate and incubate for 5 minutes at room temperature Follow Prepito truXTRAC DNA FFPE Kit instructions to purify the DNA Part Number 010295 RevA 15 Page July 2015 Patents Granted and Pending APPENDIX A EX
11. n ultrasonicator Rack Holder Rack XT 12 Place 8 Rack 12 Place 8 Rack E220e 8 microTUBE Insert microTUBE Strip V2 microTUBE Strip V2 Strip V2 PN500437 PN500485 PN500444 P Heat Block Rack E220e 8 microTUBE Strip V2 PN500493 x2 Heat Block Rack 12 Place 8 microTUBE Strip V2 PN500481 x2 Accessories Referred to as Covaris Heat Block in text Cap Press Tool 8 microTUBE Strip V2 PN 500469 Chemagen Kit Prepito truXTRAC DNA FFPE Kit CMG 2037 Optional parts FFPE tissuePICK PN 520163 FFPE sectionPICK PN 520149 FFPE sectionWARMER PN 500403 Accessories Part Number 010295 Rev A 3 Page July 2015 Patents Granted and Pending PerkinElmer chemagic Instruments and Parts e chemagic Prepito D or equivalent e Prepito truXTRAC DNA FFPE Kit CMG 2037 Other supplies e Dry block heater eg from VWR with at least two blocks capacity We recommend two dry block heaters preset at T set and T set respectively See Appendix D and E for nominal dimensions of the Covaris Heat Block and calibration procedure e RNase A DNase free at 10 mg ml e g Thermo Scientific PN ENO531 Part Number 010295 RevA 4 Page July 2015 Patents Granted and Pending PROCEDURE WORKFLOW OVERVIEW Three different protocols are supported for the Covaris truXTRAC FFPE DNA Kit The three options have different DNA extraction workflows The DNA purification workflow is identical for all three options
12. olecular biology applications This product is not intended for the diagnosis prevention or treatment of a disease INTRODUCTION The truXTRAC FFPE DNA Kit for chemagen Technology is designed for the efficient extraction of DNA from Formalin Fixed Paraffin Embedded FFPE tissue samples with Covaris Adaptive Focused Acoustics AFA and subsequent purification with PerkinElmer chemagen Technology The process results in high yields of high quality DNA well suited for analytical methods such as next generation sequencing or qPCR AFA enables effective removal of paraffin from FFPE tissue samples in aqueous buffer with simultaneous tissue rehydration Compared to chemical based methods of paraffin removal this mechanical process is not as limited by the thickness of FFPE tissue sections The ability to use thicker sections can increase DNA yield and minimize the impact of increased DNA degradation at the exposed surfaces of a section This protocol is optimized for sections up to 25 um in thickness and cores up to 1 2 mm in diameter Important Notes on FFPE Samples The yield of DNA from FFPE tissue blocks is highly variable Factors such as fixation time size and thickness of the sections the ratio of tissue to wax the type of tissue and the age of the FFPE block are the main causes for this variability The quality of DNA isolated from FFPE samples depends on many of the same factors and is thus also highly variable During the fixa
13. rbs some of too large when following OptionA Low yield for NGS library construction or by qPCR paraffin in the sample Incomplete crosslink reversal with protocol If it isn t possible to completely trim the paraffin from the FFPE block we recommend running a treatment time course by increasing the treatment time by 30 seconds steps Check that dry block heater was Calibrated following procedure in Appendix E the acoustic energy and will adversely affect DNA shearing efficiency Incomplete crosslink reversal will prevent DNA amplification Part Number 010295 RevA July 2015 Patents Granted and Pending 18 Page APPENDIX D COVARIS HEAT BLOCK NOMINAL DIMENSIONS Heat Block Rack 12 Place 8 microTUBE Strip V2 PN 500481 referred to as Covaris Heat Block in text in millimeters re 149 86 Hole for glass thermometer j 49 53 38 10 APPENDIX E DRY BLOCK HEATER CALIBRATION PROCEDURE Place the Covaris Heat Block into the dry block heater Add water to the separate hole in the Covaris Heat Block and insert a glass thermometer Set the dry block heater temperature to 60 C Wait for the dry block heater to reach the set point Check temperature displayed by the thermometer Tth If Tth is between 59 C and 61 C setpoint 1 C use 60 C for Tsety Soy OT Ro i NM Otherwise use the formula below to obtain T set T set C 120 C Tth
14. rip and remove the Cap Strip Keep 8 microTUBE 130 Strip upright while handling to avoid losing the tube insert 3 Load the 8 microTUBE 130 Strip without Cap Strip into the Cap Press Tool as shown 4 Add 100 ul Processing Buffer master mix into each microTUBE and load FFPE tissue section or core Part Number 010295 RevA 10 Page July 2015 Patents Granted and Pending NOTE if the FFPE tissue samples are loose or broken the samples may be added to the microTUBE prior to Processing Buffer addition to facilitate easier loading 5 Place the Cap Strip on top of the 8 microTUBE 130 Strip with the white side facing up 6 Close the Cap Press Tool and apply pressure until the lever bottoms out snapping the Cap Strip into place Lift the Cap Press Tool and make sure that the Cap Strip is level 7 Load 8 microTUBE 130 Strip into Rack Part Number 010295 RevA 11 Page July 2015 Patents Granted and Pending 8 Repeat steps 2 to 7 until all samples have been loaded 9 Close the Rack using the screws affixed to the cover 10 Process the sample using the settings provided in Table 3 below to dissociate the paraffin while simultaneously rehydrating the tissue Please see the example in Appendix B During the AFA process it is normal for the solution to turn milky white as the paraffin is emulsified NOTE Processed samples are stable for up to 8 hours while remaining samples are treated Ta
15. t at temperature T seth If using the same dry block heater for both the T set amp T set incubations the rack containing the samples should be stored at room temperature until the Covaris Heat Block reaches T sety Once T set has been reached load the rack containing the samples into the Covaris Heat Block 13 For Option C Extract Genomic DNA Proceed to Section 3 DNA Purification 14 For Option B Extract large DNA Fragments gt 2 kb with Improved yield process the samples using the settings in Table 4 below to release the DNA with AFA and proceed to Section 3 DNA Purification with Prepito Part Number 010295 RevA 13 Page July 2015 Patents Granted and Pending Table 4 DNA release with AFA Eege Duty Peak Incident Cycles per s Temperature Factor Power burst Instrument E220 10 105 Watts 200 10 sec 20 C E210 10 3 Intensity 200 10 sec 20 C LE220 30 300 Watts 200 10 Sec 20 C 15 For Option A Extract and Fragment DNA for NGS process the samples using the appropriate settings in Table 5 below for the desired DNA fragment size then proceed to Section 3 DNA Purification with Prepito NOTE If the target size is not achieved using the settings shown in Table 5 the treatment time should be adjusted Table 5 DNA Shearing settings E Series Focused ultrasonicator Targeted fragment size 200 bp 300 bp Treatment Time 300 sec 110 sec PIP E220 175 W 175 W Intens
16. tion process DNA is cross linked to proteins and other nucleic acid molecules to varying degrees Incomplete reversal of this crosslinking may cause the isolated DNA to perform less well in downstream enzymatic applications such as qPCR In addition the size of DNA fragments isolated from FFPE samples is generally smaller than that of DNA isolated from fresh or frozen tissues This is particularly evident in older FFPE sample blocks or sample blocks stored at elevated temperatures Note for first time users Given the highly variable yield of DNA from FFPE tissue blocks we recommend using FFPE blocks that have been well characterized for yield and quality for initial testing of the truXTRAC FFPE kit Ideally samples should be extracted immediately after sectioning Please contact Covaris at Application Support ApplicationSupport covarisinc com if you have any questions Part Number 010295 RevA 2 Page July 2015 Patents Granted and Pending REVISION HISTORY Part Number Revision Date Description of change 010295 A July 2015 Initial release K T CONTENTS Tissue SDS Buffer 10 ml PK Solution 2x 1 25 ml 8 microTUBE 130 AFA Fiber Uncapped Strip V2 12 SDS information available at http covarisinc com resources safety data sheets STORAGE This kit should be stored at room temperature 18 25 C SUPPLIED BY USERS Covaris Instruments and Parts Required parts F d i mats LE220 E220 amp E210 E220 evolutio
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