truXTRAC FFPE RNA Kit
Contents
1. 24 Place microTUBE Screw Cap PN 500308 and Intensifier PN500141 E220 evolution Rack E220e 4 Place microTUBE Screw Cap PN500432 and Intensifier PN500141 S Series Holder microTUBE Screw Cap PN500339 M220 Holder XTU PN500414 amp Insert XTU PN500489 or Holder XT PN500358 Centrifuge and Heat Block microTUBE Adapter PN500406 FFPE tissuePICK PN520163 FFPE sectionPICK PN520149 FFPE sectionWARMER PN500403 This holder has been discontinued Other supplies e Microcentrifuge with 16 000 x g capability e Dry heating block such as Eppendorf ThermoMixer or similar with either 1 5 or 2 mL heat block inserts We recommend two heating blocks preset at 56 C and 80 C respectively e Ethanol gt 96 MB Grade e g Thermo Scientific PN BP2818 100 e 1 5 mLnonstick nuclease free microfuge tubes e g Life Technologies PN AM12450 e Nuclease Free water e g Life technologies PN AM9932 or equivalent Part Number 010268 RevE September 2015 Patents Granted and Pending 4 Page 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 secti2. procedures For good yields of high quality RNA Use a maximum fixation time of 24 hours Use Formalin solution neutral buffered 4 Fix sample tissue sample as quickly as possible after collection Buffers 1 Add 275 uL of Nuclease Free water to DNase bottle Mix by inverting bottle until pellet is completely dissolved Do not vortex We recommend storing the rehydrated DNase into aliquots using sterile RNase free microcentrifuge tubes Store reconstituted DNase at 20 C 2 Add ethanol to RNA Wash Add 25 ml EtOH to 11 ml RNA Wash buffer before use 3 Check Buffer B1 and RNA Lysis Buffer A white precipitate may form during storage Incubate the bottles 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 instruments User Manual 1 ForS 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 E220 evolution Load the plate definition 500432 Rack E220e 4 Place microTUBE Screw Cap and check that the intensifier is in place 210 or E220 Load the plate definition 500308 Rack 24 Place microTUBE Screw Cap and check that the intensifier is in place LE Series load the plate definition 500388 Rack XT 24 Place microTUBE Screw Cap NOTE If you do no
3. 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 Part Number 010268 RevE 2 Page September 2015 Patents Granted and Pending REVISION HISTORY Part Number Revision Date Description of change 010268 A 11 14 Initial release 010268 B 3 15 Updated M220 Holder requirement 010268 C 5 15 Changed name of Heat Block microTUBE Adapter 010268 D 7 15 Sample input requirements clarified Changed incubation time to reverse formaldehyde 010268 E 09 15 crosslinks to 1 hour KIT CONTENTS RNA Lysis 3ml B1 Buffer 7 5 ml RNA Wash 11 ml RNA Elution 3 ml PK Solution 300 ul DNase Lyophilized 1 Vial DNase Buffer 220 ul MnCl2 Solution 400 ul RNA Purification Columns 25 RNA Collection Tubes 25 RNA Elution Tubes 25 microTUBE 130 AFA Fiber Pre Slit Screw Cap 25 SDS INFORMATION IS AVAILABLE AT http covarisinc com resources safety data sheets STORAGE This kit should be stored at room temperature 18 25 C The reconstituted DNase should be stored at 20 C Part Number 010268 RevE 3 Page September 2015 Patents Granted and Pending SUPPLIED BY USERS Covaris Instruments and Parts Focused ultrasonicator Rack Holder Insert Accessories Accessories LE220 Rack XT 24 Place microTUBE Screw Cap PN500388 E220 amp E210 Rack
4. PROTOCOL Covaris truXTRAC FFPE RNA Kit 25 Adaptive Focused Acoustics AFA based RNA extraction and purification from Formalin Fixed Paraffin Embedded Tissue Contents INTENDED USE 25 socnpcus patsy E E E E 2 INTRODUCTION EE 2 REVISION EST ee 3 KIT CONTENT TEE 3 STORAGE EE 3 S P PLEDD BV U SERS re ec 4 A A dE NE EAK EERE ee 5 FFPE Tissue Sample eesssssssnsnnnnesssssssssrerrrrrsssssssssssrrerrrrnssssssnrrerreressssseerrererer 5 Beete ege eegene 6 e Gg ln EE 6 2 RNA EXTRACTION FROM FFPE TISSUE sccsssccasscoasccessccnssccasccccssccssceascenssceaseces 8 SO RNA PURIFICATION EE 11 APPENDIX A PARAFFIN EMULSIFICATION WITH AFA ENERGY ssssssssssssssessrsrrseesessesrrseese 12 APPENDIX B TROUBLESHOOTING GUIDE sasescssscnsccsesnpensssnedenssaeeesesensdensesnbeendvanedanees 13 Part Number 010268 RevE 1 Page September 2015 Patents Granted and Pending INTENDED USE The truXTRAC FFPE RNA Kit is intended for use in molecular biology research applications This product is not intended for the diagnosis prevention or treatment of a disease INTRODUCTION The truXTRAC FFPE RNA Kit is designed for the controlled and efficient extraction of RNA from Formalin Fixed Paraffin Embedded FFPE tissue samples with the Covaris Adaptive Focused Acoustics AFA AFA enables the active removal of paraffin from FFPE tissue samples in aqueous buffer allowing simultaneous tissue rehydration Compared to tradition
5. al passive chemical based methods of paraffin removal this mechanical process is not as limited by the thickness of FFPE tissue sections The AFA process enables the use of thicker sections which can increase RNA yield and minimize the impact of increased RNA degradation at the exposed surfaces of a section The truXTRAC process results in high yields of high quality RNA well suited for analytical methods such as next generation sequencing or qPCR This protocol is optimized for sections up to 25 um in thickness Important Notes on FFPE Samples The yield of RNA 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 RNA isolated from FFPE samples is also highly variable During the fixation process RNA is cross linked to proteins and other nucleic acid molecules to varying degrees Incomplete reversal of this crosslinking may cause the isolated RNA to perform less well in downstream applications such as PCR and qPCR In addition the size of RNA fragments isolated from FFPE samples is generally smaller than that of RNA 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 RNA from FFPE tissue blocks we
6. ately and mixed fresh for each set of RNA extractions Part Number 010268 RevE 9 Page September 2015 Patents Granted and Pending 11 Add 30 ul of freshly prepared DNase master mix to each sample and mix by pipetting gently 12 Incubate for 15 minutes at room temperature 20 25 C 13 Proceed to Section 3 immediately RNA Purification Part Number 010268 RevE 10 Page September 2015 Patents Granted and Pending 3 RNA PURIFICATION 1 If DNase treatment was not conducted add 30 ul of nuclease free water to each sample 2 Add 175 ul Buffer B1 to your samples and vortex to mix 3 Add 200 ul ethanol gt 96 to samples and vortex to mix 4 Insert RNA Purification Columns into provided RNA Collection Tubes 5 Transfer the samples to the RNA Purification Columns Note Small amounts of residual wax will not interfere with the RNA purification 6 Spin the assemblies RNA Purification columns collection tubes at 14 000 x g for 1 minute NOTE If all the sample did not pass through the column please add an additional 5 minutes spin at 14 000 x g 7 Discard the flow through and place the RNA Purification Column back in the Collection Tube 8 UC wash Add 500 ul RNA Wash Spin the assemblies at 14 000 x g for 1 minute 9 Discard the flow through and place the Purification Columns back in the Collection Tubes 10 2 wash Add 500 ul RNA Wash Spin the assemblies at 14 000 x g for 1 minute 11 Discard th
7. e flow through and place the RNA Purification Columns in the RNA Collection Tubes 12 Dry RNA Purification Column Spin the assemblies at 14 000 x g for 2 minutes 13 Elute RNA RNA elution is performed in two steps Step 1 Place the RNA Purification Columns into a RNA Elution Tubes and add 20 to 50 ul RNA Elution Buffer to the center of the column Spin the assemblies at 200 x g for 2 minutes Step 2 Without removing the assemblies from the centrifuge spin at 14 000 x g for an additional 1 minute 14 Remove assemblies from the centrifuge discard RNA Purification Columns and keep the eluted RNA on ice for further analysis RNA should be kept at 80C for long term storage NOTE RNA Elution buffer composition is Nuclease Free Water Part Number 010268 RevE 11 Page September 2015 Patents Granted and Pending APPENDIX A PARAFFIN EMULSIFICATION WITH AFA ENERGY Paraffin is emulsified in 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 Additional Notes 1 Best Practices to determining the yield and purity of isolated RNA e To determine RNA yield with the highest level of accuracy a fluorometric assay such as the Qubit Life Technologies should be used e In addition spectrophotometric analysis of RNA for A260 280 and A260 230 ratios will determine if protein or peptide contamination is prese
8. gs System Duty Peak Incident Cycles per Treatment Temperature Factor Power burst Time Instrument 220 or E220 10 175 Watts 200 300 sec 20 C S2 or E210 10 5 Intensity 200 300 sec 20 C M220 20 75 Watts 200 300 sec 20 C LE220 1 15 450 Watts 200 300 sec 20 C 1 As Covaris LE220 process multiple samples at a time its PIP is distributed across microTUBEs and power received by individual microTUBEs stays within the 200 W limit Open Screw Cap microTUBE add 10 ul of PK solution to the sample and affix Screw Cap back in place Process the sample using the settings provided in Table 3 below to properly mix Proteinase K with the sample Table 3 Proteinase K mixing settings Duty Peak Incident Cyclesper Treatment Temperature Factor Power burst Time Instrument 220 or E220 10 175 Watts 200 10 sec 20 C System S2 or E210 10 5 Intensity 200 10 sec 20 C M220 20 75 Watts 200 10 sec 20 C 30 450 Watts 10 sec Part Number 010268 RevE 8 Page September 2015 Patents Granted and Pending 5 Proteinase K digestion a Insert the required number of Centrifuge and Heat Block microTUBE Screw Cap Adapters into a Heat Block b Load the microTUBEs into the adapters and incubate for 15 minutes at 56 C NOTE Some tissue types such as fibrous or muscle tissues may require longer lysis incubations to maximize RNA recovery i e 2 hours to overnight Incubation time for these types of tissues should be de
9. nt in the sample 2 See following link http covarisinc com wp content uploads pn_010268 pdf for updates to this document 3 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 010268 RevE 12 Page September 2015 Patents Granted and Pending APPENDIX B TROUBLESHOOTING GUIDE Issue Cause Solution Comments Suggestions Low yield of RNA Low tissue to wax ratio in FFPE section 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 Excess paraffin will adversely affect the yield and quality of DNA and RNA extracted from FFPE In your initial use of the truXTRAC FFPE kit use FFPE blocks that have been well characterized for yield and quality Insufficient tissue input Increase FFPE tissue section thickness or use more sections up to 5mg total weight No RNA Ethanol not added to Repeat the procedure with buffer RNA Wash fresh samples and ensure ethanol is added to RNA Wash No RNA Step 4 has been omitted Repeat the procedure with Step 4 mixes the appropriate mixing Proteinase K with the sample Proteinase K Buffer contains Glycerol and may fall to the bottom of the microTUBE Be sure to mix a
10. ons between 15 and 25 um thick NOTE 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 Size thickness 4to10 um 7 to 10 um 7 to 15 um 16 to 25 um or diameter Size length NA lt 10 mm A Collection tool tissuePICK B sectionPICK B NA Maximum 2x tissuePICK number of 200 mm tissue 2x sectionPICK 2 1 samples Per for a 5 um Tube section Numbers represent trimmed sections only NOTES A Ifthe FFPE sample is longer than about 10 mm cut it in half before loading B For optimal tissuePICK and sectionPICK performance tissue sections should be mounted on uncoated slides The tissuePICK and sectionPICK should always be used in conjunction with a sectionWARMER Part Number 010268 RevE 5 Page September 2015 Patents Granted and Pending WARNING The total mass of FFPE sample processed per extraction should be between 2 to 5 mg Lower amounts may result in insufficient yield and higher amounts may cause spin columns to become partially or fully clogged 2 Tissue Fixation Requirements The yield and quality of RNA extracted from FFPE tissue blocks is highly dependent on tissue collection and paraffin embedding
11. ppropriately RNA concentration is too low Elution volume is too high Repeat procedure using lower elution volume 20 ul minimum volume is required Alternatively Concentrate samples using ethanol precipitation or other means RNA does not perform well in downstream applications such as qPCR RNA in FFPE sample blocks is severely cross linked or degraded Design amplicons to be as small as possible lt 100 bp RNA isolated using Covaris AFA technology is of the highest possible quality Some FFPE sample blocks may be too degraded or cross linked for some applications Part Number 010268 RevE September 2015 Patents Granted and Pending 13 Page
12. t see a plate definition on your system please contact Covaris technical support at TechSupport covarisinc com Part Number 010268 RevE 6 Page September 2015 Patents Granted and Pending Table 1 Focused ultrasonicator setup Instrument Water level Fill RUN scale Chiller temperature S Series 15 18 C E Series amp L Series 10 18 C 2 For M Series Focused ultrasonicators put the Holder XTU and the Insert XTU microTUBE 130 ul or Holder XT microTUBE in place and fill the water bath until the water reaches the top of the holder Allow system to reach temperature 20 C 3 Insert the required number of Heat Block microTUBE Adapters into two heat blocks and heat them to 56 and 80 C Part Number 010268 RevE 7 Page September 2015 Patents Granted and Pending 2 RNA EXTRACTION FROM FFPE TISSUE 1 Open microTUBE Screw Cap add 110 ul RNA Lysis Buffer into microTUBE and load FFPE tissue Affix Screw Cap back in place NOTE if the FFPE tissue samples are loose or broken the samples may be added to the microTUBE prior to RNA Lysis Buffer addition to facilitate easier loading Process the samples using the settings provided in Table 2 below to dissociate the paraffin and rehydrate the tissue Please see the example in Appendix A During the AFA process it is normal for the solution to turn milky white as the paraffin is emulsified Table 2 Paraffin removal and tissue rehydration settin
13. termined empirically or contact Covaris Application Support 6 Reverse formaldehyde crosslinks a Insert the required number of Centrifuge and Heat Block microTUBE Screw Cap Adapters into a Heat block b Load the microTUBEs into the adapters and incubate for 1 hour at 80 C NOTE If you are using the same heat block for both the 56 C amp 80 C incubations the microTUBE should be stored at room temperature until the heat block reaches 80 C 7 Transfer the sample to a clean 1 5 ml microcentrifuge tube and centrifuge at 15 000 x g for 15 minutes 8 Transfer the supernatant to a new microcentrifuge tube taking care to leave the pellet and residual wax behind Small amounts of residual wax will not interfere with the RNA purification 9 Skip to Section 3 RNA Purification if DNase I treatment is not required 10 DNase treatment Optional The sample can be treated with DNase to remove residual DNA before RNA purification Prepare the DNase master mix as shown below Component Volume Per Sample in pl MnCl2 Solution DNase Buffer Total Volume per sample 30 ul NOTES DNase usage Prepare only the amount of DNase master mix required Thaw and keep the DNase enzyme on ice during use The DNase enzyme is sensitive to physical inactivation Mix by gentle pipetting Do not vortex Prepare the DNase treatment mix immediately before use The components of the DNase master mix should be stored separ
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