NucleoSpin® DNA FFPE XS - MACHEREY
Contents
1. DNA isolation from FFPE samples User manual NucleoSpin DNA FFPE XS June 2014 Rev 02 MACHEREY NAGEL www mn net com DNA Isolation from FFPE Samples Protocol at a glance Rev 02 NucleoSpin DNA FFPE XS Protocol 5 1 DNA isolation with Paraffin Dissolver Protocol 5 2 DNA isolation with xylene Sample preparation For appropriate sample For appropriate sample quantity see section 2 4 quantity see section 2 4 1 Deparaffinize sample 400 uL Paraffin Dissolver 1 mL xylene 60 C 3 min RT 2 min Mix hot sample Mix 11 000 x g 2 min Let sample cool down Discard supernatant 1 mL 98 ethanol Mix 11 000 x g 2 min Discard supernatant Dry at 60 C 3 10 min 2 Lyse sample 100 uL FL Mix vigorously 10O HERE i e gt 11 000 x g 1 min 10 uL Proteinase K 10 uL Proteinase K Mix lower phase Mix RT 3 hours or overnight RT 3 hours or overnight 3 _ Decrosslink 100 uL D Link 100 uL D Link Mix gently Mix gently eS 11 000 x g 30 s 90 C 30 min 90 C 30 min 4 Adjust binding 200 uL 98 ethanol 200 uL 98 ethanol conditions Mix Mix gt 1 000xg 1s u 5 Bind DNA Load aqueous lower phase Load Iysate R 2 eS 2 000 x g 30s 2 000 x 9 30 s 6 Wash and dry silica 1st 400 uL B5 400 uL B5 membrane e gt 11 000 x g 30 s 11 000 x g 30 s a 5 ae 400 uL B5 400 uL B5 Cc 11 000 x g 2 min 11 000 x g 2 min T Eluta DNA g 20 pL BE 20 pL BE y 11 000 x g 30 s 11 0002. Both methods show same results and effiency Deparaffinization with Paraffin Dissolver Section 5 1 Deparaffinization with xylene Section 5 2 5 1 DNA purification from FFPE samples using Paraffin Dissolver Before starting the preparation Check if Proteinase K and Buffer B5 were prepared according to section 3 Check if 96 100 ethanol is available Set incubator s at 60 C for paraffin melting and 90 C for decrosslink step Sample preparation Insert FFPE section s in microcentrifuge tube not supplied For appropriate sample amounts see section 2 4 1 Deparaffinize sample Add 400 uL Paraffin Dissolver to the sample Incubate 3 min at 60 C to melt the paraffin 400 uL Vortex the sample immediately at 60 C at a vigorous Paraffin speed to dissolve the paraffin Dissolver Cool down sample to room temperature 60 C 3 min Make sure that paraffin completely melts during the heat incubation step and mix well after melting to completely Vortex dissolve the paraffin hot sample Insufficient mixing of the heated sample may cause recurrence of solid paraffin particles Make sure the sample does not comprise more than 15 mg paraffin or adjust the volume of Paraffin Dissolver see section 2 4 MACHEREY NAGEL 06 2014 Rev 02 15 DNA isolation Paraffin Dissolver For samples comprising more than 15 mg paraffin use 30 uL Paraffin Dissolver per 1 mg paraffin If more than 400 uL Paraf
3. P 337 313 P 342 311 P 403 233 IF IN EYES Rinse continuously with water for several minutes Remove contact lenses if present and easy to do continue rinsing BEI KONTAKT MIT DEN AUGEN Einige Minuten lang behutsam mit Wasser sp len Vorhandene Kontaktlinsen nach M glichkeit entfernen Weiter aussp len Call a POISON CENTER doctor if you feel unwell Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen IF skin irritation occurs Get medical advice attention Bei Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen Get medical advice attention Bei anhaltender Augenreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTERF doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM Arzt anrufen Store in a well ventilated place Keep container tightly closed Beh lter dicht verschlossen an einem gut bel fteten Ort aufbewahren For further information please see Material Safety Data Sheets www mn net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenbl ttern www mn net com 14 MACHEREY NAGEL 06 2014 Rev 02 DNA isolation Paraffin Dissolver 5 Protocols NucleoSpin DNA FFPE XS kits offer two different methods for sample deparaffinization One utilizes the Paraffin Dissolver included in the kit and one utilizes xylene or comparable organic solvents not supplied with the kit
4. Rev 02 27 Genomic DNA from FFPE samples FORBIDDEN The respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for IN VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for IN VITRO diagnostic use Please pay attention to the package of the product IN VITRO diagnostic products are expressiy marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR IN VITRO DIAGNOSTIC USE ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE No claim or representations is intended for its use to identify any specific organism or for clinical use included but not limited to diagnostic prognostic therapeutic or blood banking It is rather in the responsibility of the user or in any case of resale of the products in the responsibility of the reseller to inspect and assure the use of the DNA RNA protein purification products of MACHEREY NAGEL for a well defined and specific application MACHEREY NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in house quality control product documentation and marketing material This MACHEREY NAGEL product is shipped with documentation stating specifica
5. The following aspects should be considered when applying one of the listed methods especially when comparing efficiency of different DNA isolation and decrosslink procedures or the usability of the isolated DNA A high DNA yield as determined by A readings or by fluorescent dye e g PicoGreen analysis does not necessarily result in good performance of the DNA in a PCR DNA may be highly degraded i e smaller fragments than the PCR target or insufficiently decrosslinked Low or no DNA yield as determined by A readings will most likely result in poor PCR results but it is still possible to achieve a good performance There may be a small amount DNA which is decrosslinked sufficiently and shows good reactivity DNA of high molecular weight does not guarantee a good amplifiability in PCR or reactivity in other enzymatic reactions DNA may be insufficiently decrosslinked although it has high molecular weight DNA of low molecular weight i e highly degraded DNA with fragment sizes exclusively below 200 nucleotides will certainly not enable amplification of fragments exceeding this size However it is still likely that small sized target sequences e g 80 150 bp can be amplified successfully especially if the DNA is well decrosslinked Neither DNA yield molecular weight absorbance ratios nor size distribution can reliably predict the performance in downstream PCR applications especially if different purification and
6. glance is designed to be used only as a supplemental tool for quick referencing while performing the purification procedure All technical literature is available on the internet at www mn net com Please contact Technical Service regarding information about changes of the current user manual compared to previous revisions MACHEREY NAGEL 06 2014 Rev 02 5 Genomic DNA from FFPE samples 2 Product description Formalin fixed paraffin embedded FFPE tissue samples are routinely prepared from human surgical tissue samples by fixation with formalin and embedding in paraffin Thin sections of FFPE samples are commonly subjected to histophathological analysis and remaining paraffin tissue blocks are usually archived Existing extensive archives of FFPE tissue samples represent a valuable source for retrospective studies of gene expression patterns and mutation analysis However the use of such samples for DNA analysis is limited due to chemical modification by formaldehyde and fragmentation of the DNA during tissue processing sampling fixing embedding and storage humidity time temperature of the samples Standard DNA isolation procedures often result in low DNA yield or poor performance in downstream applications e g PCR A special purification system taking the unique requirements of FFPE tissue into account is inevitably necessary for successful analysis of nucleic acids from FFPE samples 2 1 The basic principle The N
7. or 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze Proteinase K Proteinase K Iyophilized Danger 315 319 261 280 Proteinase K Iyophilisiert 0 Gefahr 334 335 302 352 304 340 305 351 338 312 332 313 337 313 342 311 403 233 Hazard phrases H 315 Causes skin irritation Verursacht Hautreizungen H 319 Causes serious eye irritation Verursacht schwere Augenreizung H 334 May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursa chen H 335 May cause respiratory irritation Kann die Atemwege reizen Precaution phrases P 261 Avoid breathing dust Einatmen von Staub vermeiden P 280 Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen P 302 352 IF ON SKIN Wash with plenty of water BEI KONTAKT MIT DER HAUT Mit viel Wasser waschen P 304 340 IF INHALED Remove victim to fresh air and keep at rest in a position com fortable for breathing BEI EINATMEN An die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert MACHEREY NAGEL 06 2014 Rev 02 13 Genomic DNA from FFPE samples Precaution phrases P 305 351 338 P 312 P 332 313
8. x g 30 s 8 Optional Remove u residual ethanol 90 C 8 min 90 C 8 min Y MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 52355 D ren Germany Tel 49 24 21 969 270 Fax 49 24 21 969 199 tech bio mn net com www mn net com Genomic DNA from FFPE samples Table of contents 1 Components 4 1 1 Kit contents 4 1 2 Reagents consumables and equipment to be supplied by user 5 1 3 About this user manual 5 2 Product description 6 2 1 The basic principle 6 2 2 Kit specifications 7 2 3 Handling preparation and storage of starting materials 8 2 4 Quantities of FFPE sections 8 2 5 Elution procedures 9 2 6 Stability of isolated DNA 10 2 7 Removal of residual traces of ethanol for highest sensitivity in downstream applications 10 3 Storage conditions and preparation of working solutions 12 4 Safety instructions 13 5 Protocols 15 5 1 DNA purification from FFPE samples using Paraffin Dissolver 15 5 2 DNA purification from FFPE samples with xylene deparaffinization 20 6 Appendix 24 6 1 Comments on DNA quality and quantity 24 6 2 Troubleshooting 25 6 3 Ordering information 27 6 4 Product use restriction warranty 27 MACHEREY NAGEL 11 2013 Rev 01 3 Genomic DNA from FFPE samples 1 Components 1 1 Kit contents NucleoSpin DNA FFPE XS 10 preps 50 preps 250 preps REF 740980 10 740980 50 740980 250 Paraffin Dissolver 5mL 25 mL 125 mL Lysis Buffer FL 8 mL 8 mL 8 mL Decrosslink Buffer D Link 8
9. GEL 06 2014 Rev 02 Genomic DNA from FFPE samples 2 2 Kit specifications The NucleoSpin DNA FFPE XS kit is recommended for the isolation of DNA from formalin fixed paraffin embedded FFPE tissue samples Samples are typically thin sections approx 3 20 um thickness of human or animal origin usually obtained by tissue resection or biopsy Sample amount The maximum sample size is determined by a the amount of tissue and b by the amount of paraffin NucleoSpin DNA FFPE XS is suitable for up to 5 mg tissue The amount of paraffin is limited to 15 mg when using the standard protocol with Paraffin Dissolver ca 7 sections of 10 um x 250 mm However larger amounts of paraffin samples may be processed by using either additional Paraffin Dissolver or by deparaffinization using xylene see also section 2 4 DNA yield strongly depends on the sample type quality quantity and time of storage Further measured DNA yield may vary considerably between different quantification methods Yield determined by absorption measurement at 260 nm or by a fluorescent dye e g PicoGreen may deviate from values obtained by quantification with PCR Even quantification values obtained via PCR with a short e g 80 bp and a long e g 300 bp amplicon may also differ considerably The deviation of quantification also depends on DNA size distribution as well as on efficiency of decrosslinking or extent of remaining crosslinks Please a
10. aration take one NucleoSpin DNA FFPE XS Column green ring placed in a CollectionTube 2 mL 100 uL D Link Vortex e gt 11 000 x g 30 s 90 C 30 min Vortex 1 000 x g 1s 200 uL ethanol Vortex MACHEREY NAGEL 06 2014 Rev 02 17 DNA isolation Paraffin Dissolver Pipette aqueous lower phase completely into the NucleoSpin DNA FFPE XS Column It is recommended to pipette a volume of 450 uL on the column to ensure that the complete aqueous lower phase is transferred the volume of the aqueous phase is approx 410 uL Small carry over of the organic upper phase has no negative effect on the binding procedure Centrifuge for 30 s at 2 000 x g If the solution does not flow through completely centrifuge for 30 s at 11 000 x g until the complete solution passed the column The recommended centrifugation at 2 000 x g is more efficient than centrifugation at 11 000 x g Discard Collection Tube with flow through and place the column in a new Collection Tube 2 mL Wash and dry silica membrane Add 400 uL Buffer B5 to the NucleoSpin DNA FFPE XS Column Centrifuge for 30 s at 11 000 x g Discard Collection Tube with flow through and place the column into a new Collection Tube 2 mL Add 400 uL Buffer B5 to the NucleoSpin DNA FFPE XS Column Centrifuge for 2 min at 11 000 x g to dry the membrane Discard the Collection Tube with flow through and place the column
11. d are stable up to one year Storage at lower temperatures may cause precipitation of salts Check that 96 100 ethanol is available undenaturated ethanol is preferable to adjust the binding conditions in the lysate and to prepare Wash Buffer B5 see below Before starting protocol prepare the following Proteinase K Add the indicated volume of Proteinase Buffer PB see following table or on the vial to dissolve lyophilized Proteinase K Proteinase K solution is stable at 20 C for 6 months Wash Buffer B5 Add the indicated volume of 96 100 ethanol see following table or on the bottle to Buffer B5 Concentrate Store Wash Buffer B5 at room temperature 18 25 C for up to one year NucleoSpin DNA FFPE XS 10 preps 50 preps 250 preps REF 740980 10 740980 50 740980 250 Wash Buffer B5 6 mL 12 mL 50 mL Concentrate Add 24 mL Add 48 mL Add 200 mL 96 100 ethanol 96 100 ethanol 96 100 ethanol Proteinase K 6 mg 30 mg 75 mg lyophilized Add 260 uL Add 1 35 mL Add 3 35 mL Proteinase Buffer PB Proteinase Buffer PB Proteinase Buffer PB 12 MACHEREY NAGEL 06 2014 Rev 02 Genomic DNA from FFPE samples 4 Safety instructions The following components of the NucleoSpin DNA FFPE XS kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features do not need to be labeled with H and P phrases up to 125 mL
12. decrosslinking systems are compared The major quality indicator for DNA isolated from FFPE samples is its performance in the intended downstream application 24 MACHEREY NAGEL 06 2014 Rev 02 Genomic DNA from FFPE samples 6 2 Troubleshooting Problem Possible cause and suggestions DNA is Poor sample quality degraded no Sample quality has a high impact on quality and amount of DNA obtained the DNA Reagents not applied or restored properly Kit Poor DNA quality or Reagents not properly restored Add the indicated volume of ethanol to Buffer B5 Concentrate and mix Reconstitute and store Proteinase K according to instructions given in section 3 Sample and reagents have not been mixed completely Always vortex vigorously after each reagent has been added No ethanol has been added after Iysis Binding of DNA to the silica membrane is only effective in the presence of ethanol storage Reconstitute and store Proteinase K according to instructions given in section 3 Store kit components as described in section 3 Keep bottles tightly closed in order to prevent evaporation or contamination yield lonic strength and pH influence A absorption as well as ratio Azs A280 For absorption measurement use 5 mM Tris pH 8 5 as diluent Please also see Manchester K L 1995 Value of A A ratios for measurement of purity of nucleic acids Biotechniques 19 208 209 Wilfinger W W Macke
13. erature conditions shown will reduce an elution volume of 20 uL to about 12 14 uL and will effectively remove traces of ethanol as described above e If the initial volume of elution buffer applied to the column is less than 20 uL heat incubation time should be reduced in order to avoid complete dryness If the elution 10 MACHEREY NAGEL 06 2014 Rev 02 Genomic DNA from FFPE samples volume is for example 5 uL a heat incubation of the eluate for 2 min at 80 C will adequately remove residual ethanol 25 without shaking amp 700 rpm 1400 rpm 20 Incubation time min 65 70 75 80 85 90 95 Incubation temperature C Figure 2 Removal of residual ethanol from the elution fraction by heat treatment In order to obtain highest PCR sensitivity heat incubation of the eluate is recommended Heat incubation may be performed at temperatures of 70 90 C in a heat block with or without shaking Effective conditions temperature time and shaking rate for ethanol removal can be read from the diagram an initial volume of 20 uL will evaporate to 12 14 uL during the described incubation MACHEREY NAGEL 06 2014 Rev 02 11 Genomic DNA from FFPE samples 3 Storage conditions and preparation of working solutions Attention Buffers FL contains chaotropic salts Wear gloves and goggles All kit components should be stored at room temperature 18 25 C an
14. fin Dissolver is necessary place sample in a 2 mL tube not provided Lyse sample Add 100 uL Buffer FL Vortex vigorously Centrifuge at 11 000 x g for 1 min Two phases will be formed a lower aqueous phase and an upper organic phase Tissue material will be transferred to the lower aqueous phase Optional The upper organic phase can be removed and discarded after centrifugation Pipette 10 uL Proteinase K solution directly into the lower aqueous phase Mix the aqueous phase by pipetting up and down several times Pipette only the lower aqueous phase up and down Avoid mixing lower phase and upper phase excessively Make sure that the Proteinase K is mixed well with the lysis buffer If multiple samples are processed preparation of a Buffer FL Proteinase K premix is recommended Add 110 uL of the premix to the reaction tube mix and centrifuge to achieve phase formation and to transfer the tissue into the aqueous lower phase Pipette aqueous phase up and down several times in order to disperse the tissue in the lysis buffer Incubate at room temperature for 3 hours to lyse sample tissue If residual unlysed tissue particles are visible after 3 hours add additional 10 uL Proteinase K solution and continue digestion for further 3 hours or overnight Note Yield of amplifiable DNA typically increases with elongated lysis time During this incubation step protein is digested and DNA is released i
15. g in highly concentrated DNA Elution volumes in the range of 5 30 uL are recommended the default volume is 20 uL 054 gt 2 0 a 5 0 4 v a 0 44 E rid 15 3 c f ES 03 4 2 5 5 10 gt oo 02 i fo F oO ae 014 7 0 5 20 uL 10 uL 5 ul acca Decreasing elution volume Figure 1 Correlation between elution volume and DNA concentration NucleoSpin DNA FFPE XS Columns The maximum percentage of template volume in a PCR reaction may vary depending on the robustness of the PCR system 40 template volume were tested using LightCycler PCR Roche with the DyNAmo Capillary SYBR Green qPCR Kit Finnzymes MACHEREY NAGEL 06 2014 Rev 02 9 Genomic DNA from FFPE samples 2 6 Stability of isolated DNA Due to its composition the Elution Buffer does not inhibit DNases i e it does not contain substances e g EDTA to complex divalent cations Therefore be aware not to contaminate Elution Buffer with DNase For short term DNA solution may be stored at 0 4 C and for long term storage at 20 C is recommended 2 7 Removal of residual traces of ethanol for highest sensitivity in downstream applications The default elution volume of NucleoSpin DNA FFPE XS is 20 uL The kit allows even lower elution volumes down to 5 uL to increase the DNA concentration see section 2 5 Be aware that a reduction of the 20 uL default elution volume will also increase the concentration of residual ethanol i
16. he photometer used An A value close to the background noise of the photometer will cause unexpected A A ratios 6 3 Ordering information Product REF Pack of NucleoSpin DNA FFPE XS 740980 10 50 250 10 50 250 NucleoSpin totalRNA FFPE 740982 10 50 250 10 50 250 NucleoSpin totalRNA FFPE XS 740969 10 50 250 10 50 250 NucleoSpin Tissue XS 740901 10 50 250 10 50 250 Paraffin Dissolver 740968 25 25 mL Collection Tubes 2 mL 740600 1000 Decrosslink Buffer D Link 740979 30 30 mL Visit www mn net com for more detailed product information 6 4 Product use restriction warranty NucleoSpin DNA FFPE XS kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly described in original MACHEREY NAGEL product leaflets MACHEREY NAGEL products are intended for GENERAL LABORATORY USE ONLY MACHEREY NAGEL products are suited for QUALIFIED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet of the product MACHEREY NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products in other fields of application Application on the human body is STRICTLY MACHEREY NAGEL 06 2014
17. her based upon warranty contract tort including negligence or strict liability arising in connection with 28 MACHEREY NAGEL 06 2014 Rev 02 Genomic DNA from FFPE samples the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the cor
18. into a new nuclease free microcentrifuge tube 1 5 mL not provided EB 5 e gt 2 000 x g 30 s Load aqueous lower phase 400 uL B5 11 000 x g 30 s e gt 400 uL B5 11 000 x g 2 min MACHEREY NAGEL 06 2014 Rev 02 DNA isolation Paraffin Dissolver Elute DNA Pipette 20 uL Buffer BE directly to the center of the silica membrane of the column 20 uL BE Elution volume may be varied from 5 30 uL For the correlation of elution volume DNA concentration and DNA yield see section 2 5 ed 11 000 x g 30s Centrifuge for 30 s at 11 000 x g Optional Remove residual ethanol Incubate the eluate 20 uL with open lid for 8 min at 90 C 90 C 8 min See section 2 7 for detailed information and recommendations for removal of residual ethanol MACHEREY NAGEL 06 2014 Rev 02 19 DNA isolation xylene deparaffinization 5 2 DNA purification from FFPE samples with xylene deparaffinization Before starting the preparation Check if Proteinase K and Buffer B5 were prepared according to section 3 Check if 96 100 ethanol is available Check if xylene or a similar reagent is available for deparaffinisation Set incubator s at 60 C for ethanol evaporation and 90 C for decrosslink step Sample preparation Insert FFPE section s in a microcentrifuge tube not supplied For appropriate sample amounts see section 2 4 1 Deparaffini
19. l by Buffer B5 Depending on the robustness of the used PCR system PCR might be inhibited if too much eluate is applied Use less eluate as template Store isolated DNA properly Eluted DNA should always be kept on ice for optimal stability since possible traces of DNases will degrade the isolated DNA Discrepancy between A quantification values and PCR quantification values Silica abrasion from the membrane Due to the typically low DNA content in small FFPE samples and the resulting low total amount of isolated DNA a DNA quantification via A absorption measurement is often hampered due to the low sensitivity of the absorption measurement When performing absorption measurements close to the detection limit of the photometer the measurement may be influenced by minor amounts of silica abrasion In order to prevent incorrect A quantification of small DNA amounts centrifuge the eluate for 30 s at gt 11 000 x g and take an aliquot for measurement without disturbing any sediment Alternatively use a silica abrasion insensitive RNA quantification method e g PicoGreen fluorescent dye 26 MACHEREY NAGEL 06 2014 Rev 02 Genomic DNA from FFPE samples Measurement not in the range of photometer detection limit e In order to obtain a significant A A ratio it is necessary Unexpected that the initially measured A and A values are significantly Asgo Acgo ratio above the detection limit of t
20. lity of DNA obtained from FFPE samples The procedure of tissue sampling post sampling delay before fixation fixation time embedding and storage conditions have a high impact on DNA quality and yield Starting from a paraffin embedded tissue block samples should be sectioned under clean conditions Paraffin sections may be stored at 4 C or lower for at least several weeks without observable effects on DNA yield or usability Long term storage of paraffin sections may have a negative effect on the DNA due to air oxidation Wear gloves at all times during the preparation Change gloves frequently 2 4 Quantities of FFPE sections The standard protocol section 5 1 allows the preparation of FFPE samples with approximately 15 mg ca 17 uL paraffin This corresponds to 17 sections of 10 um thickness and 100 mm area 7 sections of 10 um thickness and 250 mm area 5 sections of 10 um thickness and 325 mm area 4 sections of 10 um thickness and 400 mm area 3 sections of 10 um thickness and 575 mm area 2 sections of 10 um thickness and 840 mm area 1 section of 10 um thickness and 1680 mm area When using the standard procedure with Paraffin Dissolver Processing larger quantities is possible with protocol modifications see section 2 4 8 MACHEREY NAGEL 06 2014 Rev 02 Genomic DNA from FFPE samples Larger amounts of paraffin can be dissolved by adding a higher volume of Paraffin Dissolver REF 740968 25 to
21. lso see section 6 1 for considerations on determining DNA quality and quantity The innovative column design with a funnel shaped thrust ring and a small silica membrane area allows elution of DNA in as little as 5 80 uL Thus eluted DNA is highly concentrated and ready to use in all common downstream applications e g PCR DNA size distribution DNA isolated from formalin fixed paraffin embedded tissue shows size distribution from 50 to 5 000 bases Often short sized DNA from ca 100 300 bases predominate especially when the sample material is old However samples which were subjected to good tissue fixation embedding and storage conditions can yield DNA even larger than 5 000 bases DNA preparation time strongly depends on the sample and the required lysis time For best results lysis is performed at room temperature for at least three hours For some kinds of sample a longer lysis e g overnight will even result in remarkably higher DNA yield MACHEREY NAGEL 06 2014 Rev 02 7 Genomic DNA from FFPE samples Table 1 Kit specifications at a glance Parameter NucleoSpin DNA FFPE XS Sample material Up to 7 sections 10 um surface of 250 mm Typical yield Strongly depends on sample quality and amount Elution volume 5 30 uL Maximum loading volume 600 uL Format Mini spin column XS design 2 3 Handling preparation and storage of starting materials Many factors influence the yield and usabi
22. lushed back into the solution by pipetting Pipette solution up and down in order to homogenize sections Incubate at room temperature for 3 hours to lyse sample tissue If residual unlysed tissue particles are visible after 3 hours incubation add additional 10 uL Proteinase K solution and continue digestion for further 3 hours or overnight Note Yield of amplifiable DNA typically increases with elongated lysis time During this incubation step protein is digested and DNA is released into solution Vortex tube 5 s Convenient stopping point At this point the procedure can temporarily be stopped If pausing we recommend to store the samples at 20 C Set heating block to 90 C for subsequent decrosslink step Decrosslink Add 100 uL Decrosslink Buffer D Link to the lysate and vortex vigorously 5 s 60 C 3 10 min 100 pL FL 10 pL Proteinase K Vortex RT 3 hours 100 pL D Link Vortex MACHEREY NAGEL 06 2014 Rev 02 21 DNA isolation xylene deparaffinization Incubate at 90 C for exactly 30 min Vortex 5 s let cool down to room temperature approx 2 min If necessary spin down briefly to clear the lid approx 1 s at 1 000 x g Note This decrosslink step is necessary to remove crosslinks chemical modification caused by formalin from the DNA which is released in solution by the previous lysis step Decrosslinked DNA generally shows better performance in do
23. mL 8 mL 30 mL Wash Buter BS 6mL 12 mL 50 mL Concentrate Proteinase K lyophilized 6 mg 30 mg 75 mg Proteinase Buffer PB 1 8 mL 1 8 mL 8 mL Elution Buffer BE 13 mL 13 mL 13 mL NucleoSpin DNA FFPE XS Columns green rings plus 10 50 250 Collection Tubes Collection Tubes 2 mL 20 100 500 User manual 1 1 1 For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer BE 5 mM Tris HCl pH 8 5 4 MACHEREY NAGEL 06 2014 Rev 02 Genomic DNA from FFPE samples 1 2 Reagents consumables and equipment to be supplied by user Reagents e 96 100 ethanol undenaturated ethanol is preferable to prepare Wash Buffer B5 and to adjust binding conditions Optional for deparaffinisation without Paraffin Dissolver Xylene d Limonene mixtures of isoparaffinic hydrocarbons or similar reagents for deparaffinization Consumables 1 5 mL microcentrifuge tubes for sample lysis and DNA elution Disposable pipette tips Equipment Manual pipettors Centrifuge for microcentrifuge tubes Vortex mixer Thermal heating block adjustable to 60 C and 90 C Personal protection equipment e g lab coat gloves goggles 1 3 About this user manual It is strongly recommended that first time users of the NucleoSpin DNA FFPE XS kit read the detailed protocol sections of this user manual Experienced users however may refer to the Protocol at a glance instead The Protocol at a
24. n the eluate For the default elution volumes a heat incubation of the eluate is recommended if the eluate comprises more than 20 of the final PCR volume incubate eluate with open lid for 8 min at 90 C Inhibition of sensitive downstream reactions can be avoided by this precautional measure In this context please mind the remarks below a An incubation of the elution fraction at higher temperatures will increase signal output in PCR This is especially of importance if the template represents more than 20 of the total PCR reaction volume e g more than 4 uL eluate used as template in a PCR reaction with a total volume of 20 uL The template may represent up to 40 of the total PCR reaction volume if the eluate is incubated at 90 C for 8 min as described above b Typically 20 uL eluate will evaporate to 12 14 uL during heat incubation for 8 min at 90 C If higher final volumes are required please increase the volume of elution buffer e g from 20 uL to 30 uL c An incubation of the elution fraction for 8 min at 90 C will denature DNA If non denatured DNA is required for downstream applications other than PCR e g ligation or cloning we recommend incubating at a temperature below 80 C for a longer time as most DNA has a melting point above 80 C Suggestion incubate for 17 min at 75 C d The incubation of the eluate at higher temperatures may be adjusted according to Figure 2 The incubation time and temp
25. nto solution Vortex 5 s v 100 pL FL 11 000 x g 1 min 10 pL Proteinase K Mix by pipetting up and down lower phase RT 3 hours Vortex 5 s 16 MACHEREY NAGEL 06 2014 Rev 02 DNA isolation Paraffin Dissolver Set heating block to 90 C Convenient stopping point At this point the procedure can temporarily be stopped If pausing we recommend to store the samples at 20 C Decrosslink Add 100 uL Decrosslink Buffer D Link to the tube and vortex gently to mix Buffer D Link into the aqueous lower phase Centrifuge at 11 000xg for 30s to obtain phase formation Incubate at 90 C for exactly 30 min Vortex 5s and let cool down to room temperature approx 2 min If necessary spin down briefly to clear the lid approx 1 s at 1 000 x g Note This decrosslink step is necessary to remove the crosslinks chemical modification caused by formalin from the DNA which was released into solution by the previous lysis step Decrosslinked DNA generally shows better performance in downstream applications Adjust binding conditions Add 200 uL ethanol 96 100 to the tube and mix by vortexing 2 x 5 s Spin down briefly approx 1 s at 1 000 x g to achieve complete phase separation Note Avoid to centrifuge at much higher g force because nucleic acid might precipitate The ethanol will merge with the aqueous lower phase only Bind DNA For each prep
26. nuclease free Collection Tube 1 5 mL not provided Elute DNA Pipette 20 uL Buffer BE directly to the center of the silica membrane of the column 20 pL BE Elution volume may be varied from 5 30 uL For the correlation of elution volume DNA concentration and DNA yield see section 2 5 e gt 11 000 x g 30 s Centrifuge for 30 s at 11 000 x g Optional Remove residual ethanol Incubate the eluate 20 uL with open lid for 8 min at 90 C 90 C 8 min See section 2 7 for detailed information and recommendations for removal of residual ethanol MACHEREY NAGEL 06 2014 Rev 02 23 Genomic DNA from FFPE samples 6 Appendix 6 1 Comments on DNA quality and quantity Due to tissue fixation nucleic acids in FFPE samples are commonly fragmented and chemically modified by formaldehyde Formaldehyde modifications of DNA cannot be detected by standard quality control assays such as gel electrophoresis spectrophotometry fluorometry or microfluidics analysis However efficiency of enzymatic reactions e g PCR with chemically modified DNA is significantly decreased Affected DNA analysis methods and applications are for example Spectrophotometry e g absorption measurement A A go A280 Fluorometry e g RiboGreen Denaturing agarose gel electrophoresis Mirofluidics analysis e g Agilent 2100 Bioanalyzer BioRad s Experion Automated Electrophoresis System PCR Array analysis e g DNA microarrays
27. rectness of any of those applications Last updated 07 2010 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 24 21 969 270 tech bio mn net com Trademarks DyNAmo is a trademark of Finnzymes Oy LighCycler is a registeres trademark of a member of the Roche Group Micro Clear is a registered trademark of Micron Environmental Industries Neo Clear is a registered trademark of Merck KGaA NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH amp Co KG PicoGreen is a registered trademark of Molecular Probes Inc Roticlear is a registered trademark of CARL ROTH GmbH amp Co KG Roti is a registered trademark of CARL ROTH GmbH amp Co KG SYBR is a registered trademark of Molecular Probes Inc All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency or operation MACHEREY NAGEL 06 2014 Rev 02 29
28. the sample initially 30 uL Paraffin Dissolver per mg paraffin or by using xylene for deparaffinization as described in section 5 2 When using more than 400 uL Paraffin Dissolver per preparation it is necessary to use a collection tube larger than 1 5 mL to enable removal of the lower aqueous phase after the decrosslink step without spillage Note The NucleoSpin DNA FFPE XS standard procedure is recommended for samples containing up to 15 mg paraffin to ensure efficient dissolving of the paraffin with the indicated volume of Paraffin Dissolver and up to 5 mg tissue to avoid an overloading of the membrane Three sections of 20 mm x 25 mm area and 10 um thickness can contain up to 15 mg paraffin especially if only minor parts of the section contain tissue One section of 20 mm x 25 mm area and 10 um thickness does contain approximately 5 mg tissue if the section contains area wide tissue 2 5 Elution procedures High DNA concentration in the elution fraction is desirable for all typical downstream applications With regard to limited volumes of reaction mixtures high template concentration can be a crucial criterion Due to a large default elution volume standard kits often result in low concentrated DNA when small samples are processed Such DNA samples may even require a subsequent concentration to be suitable for the desired application NucleoSpin DNA FFPE XS kits allow efficient elution in very small volumes resultin
29. tions and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish to get an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whet
30. ucleoSpin DNA FFPE XS kit provides a convenient reliable and fast method to isolate DNA from formalin fixed paraffin embedded FFPE tissue specimen The procedure omits the use of flammable and malodorous xylene or d limonene commonly used for deparaffinization Further the procedure omits the difficult removal of organic solvent from often barely visible tissue pellets NucleoSpin DNA FFPE XS employs the odorless Paraffin Dissolver patent pending and allows efficient lysis in a convenient two phase system First the paraffin of FFPE sections is dissolved in the Paraffin Dissolver Tissue is then digested by proteinase to solubilize the fixed tissue and release DNA into solution Subsequently heat incubation with specially designed buffer effectively eliminates crosslinks from the previously released DNA After addition of ethanol the lysate is applied to the NucleoSpin DNA FFPE XS Column DNA is bound to the silica membrane Two washing steps help remove salts metabolites and macromolecular cellular components Pure DNA is finally eluted under low ionic strength conditions in a small volume 20 uL of Elution Buffer BE yielding highly concentrated DNA DNA preparation using NucleoSpin DNA FFPE XS kits can be performed at room temperature The eluate however should be treated with care because the Elution Buffer BE does not contain DNase inhibitors like EDTA To ensure DNA stability store frozen DNA at 20 C 6 MACHEREY NA
31. wnstream applications 4 Adjust binding conditions Add 200 uL ethanol 96 100 to the lysate and mix by vortexing 2 x 5s Spin down briefly to clear the lid approx 1 s at 1 000 x g 5 Bind DNA For each preparation take one NucleoSpin DNA FFPE XS Column green ring placed in a Collection Tube 2 mL Pipette lysate up and down two times before loading the lysate Load the lysate into the column Centrifuge for 30 s at 2 000 x g If the solution does not flow through completely centrifuge for 30 s at 11 000 x g until the complete solution passed the column The recommended centrifugation at 2 000 x g is more efficient than centrifugation at 11 000 x g Discard Collection Tube with flow through and place the column in a new Collection Tube 2 mL 90 C 30 min 200 uL ethanol Vortex Load lysate 2 000 x g 30s 22 MACHEREY NAGEL 06 2014 Rev 02 DNA isolation xylene deparaffinization Wash and dry silica membrane 400 pL B5 Add 400 pL Buffer B5 to the NucleoSpin DNA FFPE XS Column 11 000 x g 30s Centrifuge for 30 s at 11 000 x g Discard Collection Tube with flow through and place the column into a new Collection Tube 2 mL r Add 400 ul Buffer B5 to the NucleoSpin DNA FFPE XS Column 400 ul B5 Centrifuge for 2 min at 11 000 x g to dry the membrane 11 000 x g 2 min Discard the Collection Tube with flow through and place the column into a
32. y K and Chomczyski P 1997 Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity Biotechniques 22 474 481 Proteinase K digestion time Depending of the nature of the sample an optimal digestion time from 3 to 16 hours has to be determined empirically If residual unlysed tissue is still visible after 3 h continue the incubation for up to 16 hours After the first 3 h incubation additional Proteinase K may be added to the sample MACHEREY NAGEL 06 2014 Rev 02 25 Genomic DNA from FFPE samples Clogged NucleoSpin DNA FFPE XS Column Poor DNA quality or yield Sample material Too much starting material was used Overloading may lead to a decrease of DNA yield Reduce the quantity of sample material or use larger volumes of Paraffin Dissolver and or Lysis Buffer FL Insufficient disruption and or homogenization of starting material Perform only an overnight incubation if the tissue was not completely digested after 3 hours Suboptimal performance of DNA in downstream experiments Carry over of ethanol or salt Do not let the flow through touch the column outlet after the second wash with Buffer B5 Be sure to centrifuge at the corresponding speed for the respective time in order to remove ethanolic Buffer B5 completely Check if Buffer B5 has been equilibrated to room temperature before use Washing at lower temperatures decreases efficiency of salt remova
33. ze sample 1 mL xylene Add 1 mL xylene or alternative reagent to the sample RT Incubate at room temperature until the paraffin is 2min completely dissolved usually approx 2 min and vortex vigorously 10 s Vortex Make sure that the paraffin is completely dissolved 11 000 x g r F 2 min Centrifuge for 2 min at 11 000 x g a g S Discard Discard the supernatant by pipetting Do not remove supernatant any of the pellet 5s Vortex Centrifuge for 2 min at 11 000 x g 11 000 x g 2 min Discard supernatant Discard the supernatant by pipetting Do not remove Add 1 mL ethanol 96 100 to the pellet and vortex 1 mL ethanol any of the pellet Examples of alternatives to xylene are d Limonene e g Roti Histol Hemo De or mixtures of isoparafinnic hydrocarbons e g Roticlear Micro Clear Neo Clear 20 MACHEREY NAGEL 06 2014 Rev 02 DNA isolation xylene deparaffinization Incubate the open tube at 60 C for 3 10 min to dry the pellet It is important to evaporate all residual ethanol Residual ethanol may reduce DNA yield Lyse sample Add 100 uL Buffer FL and 10 uL Proteinase K to the pellet Vortex vigorously 5 s If multiple samples are processed preparation of a Buffer FL Proteinase K premix is recommended Add 110 uL of the premix to the pellet Centrifuge briefly approx 1 s at 1 000 x g Solid section residuals at the tube wall should be f
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