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GeneRead DNA FFPE Handbook

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2. sample prep into your laboratory workflow Sample preparation using the GlAcube follows the same steps as the manual procedure i e bind wash and elute enabling purification of high quality DNA The GlAcube is preinstalled with protocols for purification of plasmid DNA genomic DNA RNA viral nucleic acids and proteins plus DNA and RNA cleanup The range of protocols available is continually expanding and additional QIAGEN protocols can be downloaded free of charge at www giagen com MyQlAcube The GlAcube IMPORTANT Depending on the frequency of GlAcube usage additional RNase A may have to be ordered GeneRead DNA FFPE Handbook 03 2014 9 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 E Ethanol 96 100 Do not use denatured alcohol which contains other substances such as methanol or methylethylketone W 1 5 ml Safe Lock microcentrifuge tubes available from Brinkmann cat no 022363204 or Eppendorf cat no 0030 120 086 or 1 5 ml SafeSeal microcentrifuge tubes Sarstedt cat no 72 706 or 2 ml Safe Lock microcentrifuge tubes available from Brinkmann cat no 022363352 or Eppendorf cat no 0030 120 094 or 2 ml SafeSeal microcentrifuge tubes Sarstedt cat no 72 695 E Pipet and pipet
3. severely affects the performance of the GeneRead DNA FFPE Kit Preparation of buffers Preparing Buffer FTB Precipitates may form in Buffer FTB Make sure all precipitates are dissolved at 30 C Preparing Buffer AL Before starting the procedure check whether precipitate has formed in Buffer AL If necessary dissolve 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 GeneRead DNA FFPE Handbook 03 2014 11 Reconstituted Buffer AW can be stored at room temperature 15 25 C for up to 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 Tick the check box on the bottle label to indicate that ethanol has been added Reconstituted Buffer AW2 can be stored at room temperature 15 25 C for up tol year Note Before starting the procedure mix reconstituted Buffer AW2 by shaking Contains sodium azide as a preservative 12 GeneRead DNA FFPE Handbook 03 2014 Protocol Purification of DNA from FFPE Samples This protocol is for the purification of genomic DNA from formalin fixed paraffin embedded tissues ready for reliable next generation sequencing analysis Important points before starting E Perfo
4. March 2014 GeneRead DNA FFPE Handbook For purification of genomic DNA from formalin fixed paraffinembedded FFPE tissues for reliable next generation sequencing analysis Making improvements in life possible QIAGEN Sample amp Assay Technologies 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 GIAGEN sets standards in u Purification of DNA RNA and proteins M Nucleic acid and protein assays E microRNA research and RNAi E Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www qiagen com Contents Kit Contents 4 Storage 4 Intended Use Safety Information 5 Quality Control 6 Introduction 7 Principle and procedure 7 Automated purification 9 Equipment and Reagents to Be Supplied by User 10 Important Notes 11 Starting material 11 Preparation of buffers 11 Protocol Purification of DNA from FFPE Samples 13 Troubleshooting Guide 16 Ordering Information 18 GeneRead DNA FFPE Handbook 03 2014 3 Kit Contents Quick Start Protocol GeneRead DNA FFPE Kit 50 Catalog no 180134 Number of preps 50 GlAamp MinElute Columns 50 Collection Tubes 2 ml 1x50 D
5. ble laboratory detergent and water If the spilled liquid contains potentially infectious agents clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite GeneRead DNA FFPE Handbook 03 2014 5 24 hour emergency information Chemical emergency or accident assistance is available 24 hours a day from CHEMTREC USA amp Canada Tel 1 800 424 9300 Outside USA amp Canada Tel 1 703 527 3887 collect calls accepted Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of the GeneRead DNA FFPE Kit is tested against predetermined specifications to ensure consistent product quality 6 GeneRead DNA FFPE Handbook 03 2014 Introduction DNA preparation from FFPE tissue for next generation sequencing applications is associated with several challenges Yields are often limited due to the precious nature of the sample and the compromised status of the DNA Additionally artifacts introduced by fixation and embedding conditions and due to long term storage are most prevalent in sequencing results when starting with limited material One particular problem is the deamination of cytosine bases to deoxyuracil This leads to a C T conversion in sequencing reactions The GeneRead DNA FFPE Kit provides a streamlined procedure for efficient purification of high yields of DNA from small amounts of FFPE tissue sections Additionally the procedure include
6. e 15 25 C For storage for gt 1 year or if ambient temperatures often exceed 25 C we suggest storing Proteinase K at 2 8 C The remaining kit components can be stored at room temperature 15 25 C for up to 12 months without showing any reduction in performance and quality Check buffers for precipitate before use and redissolve at 30 70 C if necessary Intended Use The GeneRead DNA FFPE 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 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 GIAGEN kit and kit component sample preparation waste CAUTION DO NOT add bleach or acidic solutions directly to the Buffer AL and Buffer AW1 contain guanidine hydrochloride which can form highly reactive compounds if combined with bleach If liquid containing these buffers is spilt clean with suita
7. eparaffinization Solution 1 x 8 ml Buffer FTB 2 x 0 8 ml Buffer AL 33 ml Proteinase K 1 25 ml Buffer AW1 t concentrate 19 ml Buffer AW21 concentrate 13 ml Uracil N glycosylase 2x ml RNase Free Water 10 ml RNase A 100mg ml 14 mg Buffer ATE 12 ml CAUTION Contains a chaotropic salt Take appropriate laboratory safety measures and wear gloves when handling Not compatible with disinfectants containing bleach See Safety Information page 5 Before using for the first time add ethanol 96 100 as indicated on the bottle to obtain a working solution Contains sodium azide as a preservative Storage Uracil N glycosylase is shipped on dry ice The product should be stored immediately upon receipt at 20 C in a constant temperature freezer When the product is stored under these conditions and handled correctly performance is guaranteed until the expiration date see the quality control label on the tube GlAamp 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 GeneRead DNA FFPE Handbook 03 2014 short term storage of up to 4 weeks at room temperature 15 25 C does not affect performance The GeneRead DNA FFPE Kit contains a 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 temperatur
8. ese additional protocols have been provided by QIAGEN users for QIAGEN users These protocols have not been thoroughly tested or optimized by QIAGEN GIAGEN neither guarantees them nor warrants that they do not infringe the rights of third parties Other than expressly stated licenses GIAGEN makes no warranty that this kit and or its use s do not infringe the rights of third parties This kit and its components are licensed for one time use and may not be reused refurbished or resold GIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated 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 GIAGEN 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 2014 GIAGEN all rights reserved www qiagen com Australia techservice auQqiagen com Austria techservice at giagen com Belgium techservice bnI giagen com Brazil suportetecnico brasil giagen com Canada techservice ca giagen com China techservice cn giagen com Denmark techservice nordic giagen com Finland techservice nordic giagen com France
9. imum speed for 1 min to remove any residual liquid 22 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 lid of the QlAamp MinElute column and apply 20 40 yl Buffer ATE to the center of the membrane IMPORTANT Ensure that Buffer ATE is equilibrated to room temperature Dispense Buffer ATE onto the center of the membrane to ensure complete elution of bound DNA The volume of eluate will be up to 5 yl less than the volume of elution solution applied to the column 23 Close the lid and incubate at room temperature for 1 min Centrifuge at maximum speed 20 000 x g or14 000 rpm for 1 min Note Incubating the QlAamp MinElute column loaded with Buffer ATE for 5 min at room temperature before centrifugation generally increases DNA yield Flow through contains Buffer AL or AWT and is therefore not compatible with bleach See page 5 for safety information GeneRead DNA FFPE Handbook 03 2014 15 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
10. information see back cover or visit www giagen com Comments and suggestions Little or no DNA in the eluate a Poor quality of starting Samples that were fixed for over 20 hours or material stored for very long periods of time may contain very little usable nucleic acids Sections that were mounted on microscope slides may yield very little usable nucleic acids due to prolonged exposure to air b Low percentage Repeat the purification procedure with new ethanol used instead of samples using 96 100 ethanol Do not use 96 100 ethanol denatured ethanol as described on page 10 c Buffer AW1 or Buffer Make sure that Buffer AWT or Buffer AW2 AW2 prepared concentrates were diluted with the correct volume incorrectly of 96 100 ethanol as described on page 10 d DNA still bound to spin Repeat the elution step but incubate the QlAamp column MinElute spin column on the benchtop for 10 min with Buffer ATE before centrifuging Inefficient removal of deaminated cytosine a Too much starting Since the GeneRead DNA FFPE Kit is based on material an enzymatic digestion too much starting material will lead to inefficiency Reduce the amount of starting material b UNG reaction mixture Be sure to properly prepare the reaction mix by prepared incorrectly precise addition of all components and transfer of the aqueous phase in steps 6 and 11 16 GeneRead DNA FFPE Handbook 03 2014 Comments and suggestions Clogged QlAamp MinElute spin c
11. nquire 18 GeneRead DNA FFPE Handbook 03 2014 Product Contents Cat no Starter Pack QlAcube Pack includes reagent bottle racks 990395 3 rack labeling strips 8 200 pl filter tips 1024 1000 yl filter tips 1024 1000 pl filter tips wide bore 1024 30 ml reagent bottles 18 rotor adapters 120 rotor adapter holder 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 or your local distributor US Canada and Japan GeneRead DNA FFPE Handbook 03 2014 19 Trademarks QIAGEN QlAamp GlAcube GeneRead Making improvements in life possible MinElute QIAGEN Group Limited License Agreement for the GeneRead DNA FFPE Kit Use of this product signifies the agreement of any purchaser or user of the product to the following terms 1 Qr des DD 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 GIAGEN grants no license under any of its intellectual property to 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 giagen com Some of th
12. o remove drops from inside the lid 14 Add 2 pl RNase A 100 mg ml mix and incubate for 2 min at room temperature 15 Add 250 pl Buffer AL to the sample and mix thoroughly by vortexing Then add 250 pl ethanol 96 100 to the sample and mix again thoroughly by vortexing Centrifuge briefly to remove drops from inside the lid 16 Transfer 700 yl lysate to the QlAamp MinElute column in a 2 ml collection tube without wetting the rim close the lid and centrifuge at maximum speed for 1 min Discard the flow through and reuse the collection tube If the lysate has not completely passed through the membrane after centrifugation centrifuge again at a higher speed until the QlAamp MinElute column is empty 17 Repeat step 16 until the complete lysate is used Flow through contains Buffer AL or AW1 and is therefore not compatible with bleach See page 5 for safety information 14 GeneRead DNA FFPE Handbook 03 2014 18 Add 500 pl Buffer AW1 to the spin column and centrifuge at maximum speed for 1 min Discard the flow through and reuse the collection tube 19 Add 500 pl Buffer AW2 to the spin column and centrifuge at maximum speed for 1 min Discard the flow through and reuse the collection tube 20 Add 250 pl ethanol 96 100 to the spin column and centrifuge at maximum speed for 1 min Discard the flow through and the collection tube 21 Place the spin column into a new 2 ml collection tube supplied and centrifuge at max
13. olumn Centrifugation The centrifugation temperature should be 15 temperature too low 25 C Some centrifuges may cool to below 15 C even when set at 20 C This can cause formation of precipitates that can clog the QlAamp MinElute spin column If this happens set the centrifugation temperature to 25 C Warm the ethanol containing sample to 37 C before transferring it to the QlAamp mini spin column GeneRead DNA FFPE Handbook 03 2014 17 Ordering Information Product Contents Cat no GeneRead DNA FFPE For 50 preps GlAamp MinElute 180134 Kit 50 Columns Collection Tubes Deparaffinization Solution Uracil N glycosylase RNase Free Water RNase A and Buffers Accessories and reagents Deparaffinization 2 x 8 ml deparaffinization solution 19093 Solution 16 ml QIAGEN Proteinase K 2 ml 600 mAU ml solution 19131 2 ml Collection Tubes 2 ml 1000 Collection Tubes 2 ml 19201 RNase A 17 500 U 2 5 ml 100 mg ml 7000 units ml 19101 solution GlAcube for fully automated sample preparation using GIAGEN spin column kits GlAcube 110 V Robotic workstation for automated 900129 GlAcube 230 V purification of nucleic acids or 9001293 proteins using QIAGEN spin column kits 1 year warranty on parts and labor Fully automatable on the QlAcube See www giagen com MyQlAcube for protocols t US Canada and Japan Rest of world Agreements for comprehensive service coverage are available please i
14. rm all centrifugation steps at room temperature 15 25 C M Read Important Notes page 11 Things to do before starting u Equilibrate all buffers to room temperature 15 25 C E Seta thermomixer or heating block to 56 C for use in step 5 and 8 If possible preheat a second thermomixer or heating block to 90 C for use in step 9 and a third to 50 C for use in step 12 E Precipitates may form in Buffer FTB Make sure all precipitates are dissolved at 30 C W If Buffer AL contains precipitates dissolve by heating to 70 C with gentle agitation E Deparaffinization Solution solidifies at temperatures below 18 C Incubate at 30 C to liquefy E Before starting the procedure mix reconstituted Buffer AW1 and AW2 by shaking Procedure 1 Using a scalpel trim excess paraffin off the sample block 2 Cut one section up to 10 pm thick see Starting material page 11 If the sample surface has been exposed to air discard the first 2 3 sections 3 Immediately place the section in a 1 5 ml or 2 ml microcentrifuge tube not supplied 4 Add 160 pl Deparaffinization Solution vortex vigorously for 10 s and centrifuge briefly to bring the sample to the bottom of the tube 5 Incubate at 56 C for 3 min and then allow to cool at room temperature If too litle Deparaffinization Solution is used or if too much paraffin is carried over with the sample Deparaffinization Solution may become waxy or solid after cooling If
15. s the removal of deaminated cytosine to prevent false results in DNA sequencing Principle and procedure The GeneRead DNA FFPE procedure removes paraffin and reverses formalin cross links from the DNA sample before it is bound to the QlAamp MinElute column After heating to remove cross links the DNA is accessible for the specific removal of deaminated cytosine residues by the enzyme Uracil N Glycosilase UNG The optimized reaction mixture provides conditions in which the UNG can specifically remove artificially induced uracils from the DNA obtained from the FFPE sample After the binding of DNA to the spin column residual contaminants such as salts are washed away by Buffers AW and AW2 and ethanol Any residual ethanol which may interfere with subsequent enzymatic reactions is removed by an additional centrifugation step DNA is eluted and is now ready to use in next generation sequencing workflows Alternatively it can be stored at 20 C GeneRead DNA FFPE Handbook 03 2014 7 GeneRead DNA FFPE procedure y Remove paroffin D Heat Digest artifacts Bind DNA f mit 4 mit lt 9 m DNA ready to uze for reboble NGS ee EEE 8 GeneRead DNA FFPE Handbook 03 2014 Automated purification The GeneRead DNA FFPE Kit can be automated on the QlAcube The innovative QlAcube uses advanced technology to process QIAGEN spin columns enabling seamless integration of automated low throughput
16. this occurs add additional Deparaffinization Solution and repeat the 56 C incubation 6 Add 55 pl RNase free water 25 pl Buffer FTB and 20 yl proteinase K GeneRead DNA FFPE Handbook 03 2014 13 Note A master mix comprising RNase free water Buffer FTB and proteinase K may be prepared in advance 7 Vortex and briefly centrifuge the samples Note Deparaffinization Solution will form a layer above Buffer FTB with the addition of proteinase K 8 Incubate at 56 C for 1 h Note If using only one thermomixer or heating block leave the sample at room temperature 15 25 C after the 56 C incubation in step 8 until the thermomixer or heating block has reached 90 C for step 9 9 Incubate at 90 C for 1 h Incubation at 90 C in Buffer FTB partially reverses formaldehyde modification of nucleic acids Longer incubation times or higher incubation temperatures may result in more fragmented DNA Note If using only one thermomixer or heating block leave the sample at room temperature 15 25 C after the 90 C incubation in step 9 until the thermomixer or heating block has reached 50 C for step 12 10 Briefly centrifuge the tube to remove drops from inside the lid 11 Transfer the lower clear phase into a new microcentrifuge tube not provided add 115 pl RNase free water and mix 12 Add 35 pl UNG to the sample vortex and incubate at 50 C for one hour in a thermomixer or heating block 13 Briefly centrifuge the tube t
17. tips to avoid cross contamination we recommend pipet tips with aerosol barriers E Thermomixer or heating block capable of incubation at 90 C M Microcentrifuge with rotor for 2 ml tubes B Vortexer This is not a complete list of suppliers and does not include many important vendors of biological supplies SS S S K K _ gt gt K KH___IZTm N El E qy gt gt yyyylyyyE i A 10 GeneRead DNA FFPE Handbook 03 2014 Important Notes Starting material Standard formalin fixation and paraffinembedding procedures always result in significant fragmentation of nucleic acids To limit the extent of DNA fragmentation be sure to u Fix tissue 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 Thoroughly dehydrate samples prior to embedding residual formalin can inhibit proteinase K digestion Starting material for DNA purification should be a freshly cut section of FFPE tissue with a thickness of up to 10 pm Usually DNA yields from this amount of material exceed 0 5 pg which is enough for NGS downstream applications However DNA yield from FFPE samples varies greatly depending on the tissue type as well as fixation and embedding conditions Always analyze yields precisely before proceeding Avoid using too much starting material as this

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