QIAamp® DNA FFPE Tissue Handbook
Contents
1. or your local distributor Other kit sizes are available see www qiagen com QlAamp DNA FFPE Tissue Handbook 06 2012 19 Notes 20 QlAamp DNA FFPE Tissue Handbook 06 2012 Notes QlAamp DNA FFPE Tissue Handbook 06 2012 21 Notes 22 QlAamp DNA FFPE Tissue Handbook 06 2012 Trademarks QIAGEN QlAamp MinElute Qproteome REPLI g RNeasy QIAGEN Group Limited License Agreement for QlAamp DNA FFPE Tissue Kit Use of this product signifies the agreement of any purchaser or user of the product to the following terms 1 The product may be used solely in accordance with the protocols provided with the product and this handbook and for use with components contained in the kit only QIAGEN grants no license under any of its intellectual property fo use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the protocols provided with the product this handbook and additional protocols available at www qiagen com Some of these additional protocols have been provided by QIAGEN users for QIAGEN users These protocols have not been thoroughly tested or optimized by QIAGEN QIAGEN neither guarantees them nor warrants that they do not infringe the rights of third parties 2 Other than expressly stated licenses QIAGEN makes no warranty that this kit and or its use s do not infringe the rights of third parties 3 This kit and its components are licensed for on2. Salt and ethanol components of wash Buffers after storage AW1 and AW2 may have separated out after being unused for a long period Always mix buffers thoroughly before each purification procedure d Ethanol carryover Be sure to centrifuge at full speed using a new collection tube to completely dry the membrane before elution of DNA General handling Clogged QlAamp Incomplete lysis caused clogging of the MinElute column membrane Increase the lysis time to fully lyse the sample 16 QlAamp DNA FFPE Tissue Handbook 06 2012 Appendix Working with DNA General handling Proper microbiological aseptic technique should always be used when working with small sample sizes Hands and dust particles may carry bacteria and molds and are the most common sources of contamination Always wear latex or vinyl gloves while handling reagents and samples to prevent contamination from the surface of the skin or from dusty laboratory equipment Change gloves frequently and keep tubes closed Disposable plasticware The use of sterile disposable polypropylene tubes is recommended throughout the purification procedure These tubes are generally DNase free QlAamp DNA FFPE Tissue Handbook 06 2012 17 Ordering Information Product Contents Cat no QlAamp DNA FFPE For 50 DNA preps 50 QlAamp 56404 Tissue Kit 50 MinElute Columns Proteinase K Buffers Collection Tubes 2 ml Accessories RNase A 17 500 U 2 5 ml 100 mg ml 7000 units ml 19101 s
3. at 2 8 C For periods longer than 24 hours we recommend storage at 20 C For whole genome amplification WGA of DNA purified from FFPE tissues we recommend using the REPLI g FFPE Kit which is optimized for use with this DNA See page 18 for ordering information Handling of QlAamp MinElute columns Due to the sensitivity of nucleic acid amplification technologies the following precautions are necessary when handling QlAamp MinElute columns to avoid cross contamination between sample preparations Carefully apply the sample or solution to the QlAamp MinElute column Pipet the sample into the QlAamp MinElute column without wetting the rim of the column m Always change pipet tips between liquid transfers We recommend the use of aerosol barrier pipet tips Avoid touching the QlAamp MinElute column membrane with the pipet tip After all pulse vortexing steps briefly centrifuge the microcentrifuge tubes to remove drops from the inside of the lids HM Open only one QlAamp MinElute column at a time and take care to avoid generating aerosols IN Wear gloves throughout the entire procedure In case of contact between gloves and sample change gloves immediately Centrifugation QlAamp MinElute columns will fit into most standard 1 5 2 ml microcentrifuge tubes Additional 2 ml collection tubes are available separately Centrifugation of QlAamp MinElute columns is performed at 6000 x g 8000 rpm to reduce centrifug
4. 2 Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate safety data sheets SDSs These are available online in convenient and compact PDF format at www giagen com safety where you can find view and print the SDS for each QIAGEN kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste Buffer AL and Buffer AW1 contain guanidine hydrochloride which can form highly reactive compounds when combined with bleach If liquid containing this buffer is spilt clean with suitable laboratory detergent and water If the spilt liquid contains potentially infectious agents clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 613 1 19240 Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of the QlAamp DNA FFPE Tissue Kit is tested against predetermined specifications to ensure consistent product quality QlAamp DNA FFPE Tissue Handbook 06 2012 5 Introduction The QlAamp DNA FFPE Tissue Kit is optimized for purification of DNA from FFPE tissue sections lt uses well establishe
5. June 2012 QlAamp DNA FFPE Tissue Handbook For purification of genomic DNA from formalin fixed paraffin embedded tissues QIAGEN QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com Contents Kit Contents Storage Intended Use Safety Information Quality Control Introduction Principle and procedure Equipment and Reagents to Be Supplied by User Important Notes Starting material Copurification of RNA Eluting pure DNA Handling of QlAamp MinElute columns 0000000000 i Preparation of buffers Protocol E Isolation of Genomic DNA from FFPE Tissue Sections a N Troubleshooting Guide Appendix Working with DNA Ordering Information CON QlAamp DNA FFPE Tissue Handbook 06 2012 3 Kit Contents QlAamp DNA FFPE Tissue Kit 50 Catalog no 56404 Number of preps 50 QlAamp MinElute Columns 50 Collection Tubes 2 ml 3x50 Buffer ATL 14 ml Buffer AL 12 ml Buffer AW1 concentr
6. Using a scalpel trim excess paraffin off the sample block 2 Cut up to 8 sections 5 10 pm thick see Starting material If the sample surface has been exposed to air discard the first 2 3 sections 3 Immediately place the sections in a 1 5 or 2 ml microcentrifuge tube not supplied and 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 5 Remove the supernatant by pipetting Do not remove any of the pellet 6 Add 1 ml ethanol 96 100 to the pellet and mix by vortexing The ethanol extracts residual xylene from the sample 7 Centrifuge at full speed for 2 min at room temperature 8 Remove the supernatant by pipetting Do not remove any of the pellet Carefully remove any residual ethanol using a fine pipet tip 9 Open the tube and incubate at room temperature or up to 37 C Incubate for 10 min or until all residual ethanol has evaporated 10 Resuspend the pellet in 180 pl Buffer ATL Add 20 pl proteinase K and mix by vortexing 11 Incubate at 56 C for 1 h or until the sample has been completely lysed 12 QlAamp DNA FFPE Tissue Handbook 06 2012 12 13 14 15 16 17 18 Incubate at 90 C for 1 h The incubation at 90 C in Buffer ATL partially reverses formaldehyde modification of nucleic acids Longer incubation times or higher incubation temperatures may result in more fragmented DNA If using only
7. ate 19 ml Buffer AW2 concentrate 13 ml Buffer ATE 20 ml Proteinase K 1 25 ml Selection Guide 1 Contains a guanidine salt Not compatible with disinfectants containing bleach See page 5 for safety information Contains sodium azide as a preservative Storage QlAamp MinElute columns should be stored at 2 8 C upon arrival and are stable under these conditions for at least one year after delivery However short term storage of up to 4 weeks at room temperature 15 25 C does not affect performance All buffers can be stored at room temperature and are stable for at least one year after delivery The QlAamp DNA FFPE Tissue Kit contains a novel ready to use proteinase K solution which is supplied in a specially formulated storage buffer Proteinase K is stable for at least one year after delivery when stored at room temperature For storage longer than one year or if ambient temperatures often exceed 25 C we suggest storing proteinase K at 2 8 C Intended Use The QlAamp DNA FFPE Tissue Kit is intended for molecular biology applications This product is not intended for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines 4 QlAamp DNA FFPE Tissue Handbook 06 201
8. d QlAamp DNA Micro technology for purification of genomic and mitochondrial DNA from small sample volumes or sizes The kit combines the selective binding properties of a silica based membrane with flexible elution volumes of between 20 and 100 pl Specially optimized lysis conditions allow genomic DNA to be efficiently purified from FFPE tissue sections without the need for overnight incubation Incubation at an elevated temperature after proteinase K digestion partially removes formalin crosslinking of the released DNA improving yields as well as DNA performance in downstream assays Note that DNA isolated from FFPE samples is usually of lower molecular weight than DNA from fresh or frozen samples The degree of fragmentation depends on the type and age of the sample and the conditions used for fixation After sample lysis the simple QlAamp DNA Micro procedure which is highly suited for simultaneous processing of multiple samples yields pure DNA in less than 30 minutes DNA is eluted in Buffer ATE or water and is immediately ready for use in amplification reactions or for storage at 20 C Purified DNA is free of proteins nucleases and other impurities Principle and procedure The QlAamp DNA FFPE Tissue procedure consists of 6 steps see flowchart Remove paraftin paraffin is dissolved in xylene and removed Lyse sample is lysed under denaturing conditions with proteinase K Heat incubation at 90 C reverses formalin crosslinking Bi
9. e noise Centrifugation at full speed will not improve DNA yields However centrifugation of QlAamp MinElute columns at full speed is required in 2 steps of the procedure the dry centrifugation step after the membranes are washed and the elution step Centrifugation at full speed is also required to bring down the sample after the xylene treatment and the ethanol wash step All centrifugation steps should be carried out at room temperature 15 25 C 10 QlAamp DNA FFPE Tissue Handbook 06 2012 Processing QlAamp MinElute columns in a microcentrifuge m Always close QlAamp MinElute columns before placing them in the microcentrifuge Centrifuge as described in the protocol Flow through fractions may contain hazardous waste and should be disposed of appropriately IN For efficient parallel processing of multiple samples we recommend filling a rack with collection tubes into which QlAamp MinElute columns can be transferred after centrifugation Used collection tubes containing flow through can be discarded and the new collection tubes containing the QlAamp MinElute columns can be placed directly in the microcentrifuge Preparation of buffers Preparing Buffer ATL Before starting the procedure check whether precipitate has formed in Buffer ATL If necessary dissolve by heating to 70 C with gentle agitation Preparing Buffer AL Before starting the procedure check whether precipitate has formed in Buffer AL If necessary dissol
10. e time use and may not be reused refurbished or resold 4 QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated 5 The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and or its components For updated license terms see www qiagen com 2007 2012 QIAGEN all rights reserved www giagen com Australia techservice au giagen com Austria techservice at qiagen com Belgium techservice bni qiagen com Brazil suportetecnico brasil giagen com Canada techservice ca qiagen com China techservice cn qiagen com Denmark techservice nordic giagen com Finland techservice nordic qiagen com France techservice fr qiagen com Germany techservice de qiagen com Hong Kong techservice hk qiagen com India techservice india qiagen com Ireland techservice uk qiagen com Italy techservice it giagen com Japan techservice jp giagen com Korea South techservice kr qiagen com Luxembourg techservice bni qiagen com Mexico techservice mx qiagen com The Netherlands techservice bnl giagen com Norway t
11. echservice nordic qiagen com Singapore techservice sg qiagen com Sweden techservice nordic qiagen com Switzerland techservice ch qiagen com UK techservice uk giagen com USA techservice us qiagen com 1072907 06 2012 QIAGEN Sample amp Assay Technologies
12. einase K was stored at high temperatures for a prolonged time Repeat the procedure using new samples and fresh proteinase K Make sure that the samples were thoroughly dehydrated prior to embedding Residual formalin can inhibit the proteinase K digest b Low percentage ethanol used Repeat the purification procedure with new instead of 96 100 ethanol samples using 96 100 ethanol Do not use denatured alcohol which contains other substances such as methanol or methylethylketone c Buffer AW1 or Buffer AW2 Make sure that the Buffer AW1 and Buffer prepared incorrectly AW2 concentrates were diluted with the correct volume of 96 100 ethanol as described on page 11 DNA does not perform well in downstream enzymatic reactions a DNA fragmented or blocked Although the 90 C incubation in the QlAamp due to formaldehyde modification DNA FFPE Tissue procedure removes most of the formaldehyde modifications DNA purified from FFPE sections may not perform as well in enzymatic reactions as DNA from fresh or frozen samples We recommend keeping amplicons as short as possible for PCR lt 500 nucleotides b Reduced sensitivity Determine the maximum volume of eluate suitable for your amplification reaction Adjust the volume of eluate added to the amplification reaction accordingly The elution volume can be adjusted proportionally QlAamp DNA FFPE Tissue Handbook 06 2012 15 Comments and suggestions c Wash buffers not mixed well
13. he protocol The protocol describes the use of a 100 mg ml RNase A stock solution For efficient purification of RNA from FFPE tissues we recommend using the RNeasy FFPE Kit which is optimized for high yields of usable RNA from these samples See page 19 for ordering information Eluting pure DNA For downstream applications that require small starting volumes e g some PCR assays a more concentrated eluate may increase assay sensitivity QlAamp MinElute columns allow a minimum elution volume of 20 pl for concentrated nucleic acid eluates For downstream applications that require a larger starting volume the elution volume can be increased to 100 pl However an increase in elution volume will decrease the concentration of DNA in the eluate The volume of eluate recovered may be up to 5 pl less than the volume of Buffer ATE applied to the QlAamp MinElute column For example an elution volume of 20 pl results in 215 pl eluate The volume of eluate recovered depends on the nature of the sample QlAamp DNA FFPE Tissue Handbook 06 2012 9 Buffer ATE should be equilibrated to room temperature 15 25 C before it is applied to the QlAamp MinElute column Yields will be increased if the column is incubated with Buffer ATE at room temperature for 5 minutes before centrifugation Eluted DNA can be collected in standard 1 5 ml microcentrifuge tubes not provided If the purified DNA is to be stored for up to 24 hours we recommend storage
14. id of the QlAamp MinElute column and apply 20 100 pl Buffer ATE to the center of the membrane Important Ensure that Buffer ATE is equilibrated to room temperature If using small elution volumes lt 50 pl dispense Buffer ATE onto the center of the membrane to ensure complete elution of bound DNA QlAamp MinElute columns provide flexibility in the choice of elution volume Choose a volume according to the requirements of the downstream application The volume of eluate will be up to 5 pl less than the volume of elution solution applied to the column 21 Close the lid and incubate at room temperature for 1 min Centrifuge at full speed 20 000 x g 14 000 rpm for 1 min Incubating the QlAamp MinElute column loaded with Buffer ATE for 5 min at room temperature before centrifugation generally increases DNA yield 14 QlAamp DNA FFPE Tissue Handbook 06 2012 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www giagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www qiagen com Comments and suggestions Little or no DNA in the eluate a Insufficient sample lysis Prot
15. nd DNA binds to the membrane and contaminants flow through Wash residual contaminants are washed away Elute pure concentrated DNA is eluted from the membrane 6 QlAamp DNA FFPE Tissue Handbook 06 2012 QlAamp DNA FFPE Tissue Procedure Sample V V L lt L Remove paraffin Lyse Heat Bind DNA Wash Elute Ready to use DNA QlAamp DNA FFPE Tissue Handbook 06 2012 7 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate safety data sheets SDSs available from the product supplier m Xylene E Ethanol 96 100 M 1 5 ml or 2 ml microcentrifuge tubes for lysis steps a 1 5 ml microcentrifuge tubes for elution steps available from Brinkmann Safe Lock cat no 022363204 Eppendorf Safe Lock cat no 0030 120 086 or Sarstedt Safety Cap cat no 72 690 E Pipet tips to avoid cross contamination we recommend pipet tips with aerosol barriers IN Thermomixer heated orbital incubator heating block or water bath capable of incubation at 90 C Microcentrifuge with rotor for 2 ml tubes HE Vortexer E Optional RNase A 100 mg ml cat no 19101 Do not use denatured alcohol which contains other substances such as methanol or methylethylketone t This is not a complete list of suppliers and does not include many impo
16. ntil the QlAamp MinElute column is empty Carefully open the QlAamp MinElute column and add 500 pl Buffer AW1 without wetting the rim Close the lid and centrifuge at 6000 x g 8000 rpm for 1 min Place the QlAamp MinElute column in a clean 2 ml collection tube and discard the collection tube containing the flow through Carefully open the QlAamp MinElute column and add 500 pl Buffer AW2 without wetting the rim Close the lid and centrifuge at 6000 x g 8000 rpm for 1 min Place the QlAamp MinElute column in a clean 2 ml collection tube and discard the collection tube containing the flow through Contact between the QlAamp MinElute column and the flow through should be avoided Some centrifuge rotors may vibrate upon deceleration resulting in the flow through which contains ethanol coming into contact with the QlAamp MinElute column Take care when removing the QlAamp MinElute column and collection tube from the rotor so that flow through does not come into contact with the QlAamp MinElute column QlAamp DNA FFPE Tissue Handbook 06 2012 13 004044 19 Centrifuge at full speed 20 000 x g 14 000 rpm for 3 min to dry the membrane completely This step is necessary since ethanol carryover into the eluate may interfere with some downstream applications 20 Place the QlAamp MinElute column in a clean 1 5 ml microcentrifuge tube not provided and discard the collection tube containing the flow through Carefully open the l
17. olution Buffer ATL 200 ml 200 ml Tissue Lysis Buffer 19076 Buffer AL 216 ml 216 ml lysis Buffer AL 19075 Buffer AW 1 242 ml Wash Buffer 1 Concentrate 19081 concentrate 242 ml Buffer AW2 324 ml Wash Buffer 2 Concentrate 19072 concentrate 324 ml Collection Tubes 2 ml 1000 Collection Tubes 2 ml 19201 Related products RNeasy FFPE Kit for purification of high yields of usable RNA from FFPE tissues RNeasy FFPE Kit 50 For 50 RNA preps 50 RNeasy 74404 MinElute Spin Columns 50 gDNA Eliminator Mini Spin Columns Collection Tubes RNase Free Reagents and Buffers Qproteome FFPE Tissue Kit for isolation of full length proteins from FFPE tissues Qproteome FFPE Tissue Kit 20 For 20 protein preparations from 37623 formalin fixed paraffin embedded tissue samples Extraction Buffer Collection Tubes Collection Tube Sealing Clips Other kit sizes are available see www giagen com 18 QlAamp DNA FFPE Tissue Handbook 06 2012 Ordering Information Product Contents Cat no REPLI g FFPE Kit for whole genome amplification of DNA from FFPE tissues REPLI g FFPE Kit 25 DNA Polymerase Buffers and 150243 Reagents for 25 x 50 pl whole genome amplification reactions For up to date licensing information and productspecific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services
18. one heating block leave the sample at room temperature after the 56 C incubation until the heating block has reached 90 C Briefly centrifuge the 1 5 ml tube to remove drops from the inside of the lid If RNA free genomic DNA is required add 2 pl RNase A 100 mg ml and incubate for 2 min at room temperature before continuing with step 14 Allow the sample to cool to room temperature before adding RNase A Add 200 pl Buffer AL to the sample and mix thoroughly by vortexing Then add 200 pl ethanol 96 100 and mix again thoroughly by vortexing It is essential that the sample Buffer AL and ethanol are mixed immediately and thoroughly by vortexing or pipetting to yield a homogeneous solution Buffer AL and ethanol can be premixed and added together in one step to save time when processing multiple samples A white precipitate may form on addition of Buffer AL and ethanol This precipitate does not interfere with the QlAamp procedure Briefly centrifuge the 1 5 ml tube to remove drops from the inside of the lid Carefully transfer the entire lysate to the QlAamp 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 QlAamp 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 u
19. rtant vendors of biological supplies p pp y Imp 9 pP 8 QlAamp DNA FFPE Tissue Handbook 06 2012 Important Notes Starting material Standard formalinfixation and paraffinembedding procedures always result in significant fragmentation of nucleic acids To limit the extent of DNA fragmentation be sure to HE Fixtissue samples in 4 10 formalin as quickly as possible after surgical removal E Use a fixation time of 14 24 hours longer fixation times lead to more severe DNA fragmentation resulting in poor performance in downstream assays m Thoroughly dehydrate samples prior to embedding residual formalin can inhibit the proteinase K digest Starting material for DNA purification should be freshly cut sections of FFPE tissue each with a thickness of up to 10 pm Up to 8 sections each with a thickness of up to 10 pm and a surface area of up to 250 mm can be combined in one preparation If you have no information about the nature of your starting material we recommend starting with no more than 3 sections per preparation Depending on DNA yield and purity it may be possible to use up to 8 sections in subsequent preparations Copurification of RNA Using the QlAamp DNA FFPE Tissue Kit RNA may be copurified with the DNA if it is present in the sample RNA may inhibit some downstream enzymatic reactions although it does not affect PCR If RNA free genomic DNA is required RNase A should be added to the sample as indicated in t
20. ve by heating to 70 C with gentle agitation Preparing Buffer AW1 Add 25 ml ethanol 96 100 to the bottle containing 19 ml Buffer AW1 concentrate Tick the check box on the bottle label to indicate that ethanol has been added Reconstituted Buffer AW1 can be stored at room temperature 15 25 C for up to 1 year Note Before starting the procedure mix reconstituted Buffer AW1 by shaking Preparing Buffer AW2 Add 30 ml ethanol 96 100 to the bottle containing 13 ml Buffer AW2 concentrate Reconstituted Buffer AW2 can be stored at room temperature 15 25 C for up to 1 year Note Before starting the procedure mix reconstituted Buffer AW2 by shaking QlAamp DNA FFPE Tissue Handbook 06 2012 11 v e 6 he a Protocol Isolation of Genomic DNA from FFPE Tissue Sections Important points before starting l Perform all centrifugation steps at room temperature 15 25 C E Read Important Notes pages 9 11 Things to do before starting MH Equilibrate all buffers to room temperature 15 25 C E Seta thermomixer or heated orbital incubator to 56 C for use in step 11 If a thermomixer or heated orbital incubator is not available a heating block or water bath can be used instead E If Buffer AL or Buffer ATL contain precipitates dissolve by heating to 70 C with gentle agitation E Ensure that Buffer AW1 and Buffer AW2 have been prepared according to the instructions on page 11 Procedure 1
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