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NucleoSpin® FFPE RNA/DNA

1. 2 4 Quantities of FFPE sections Following the procedures of section 5 1 or 5 2 utilizing 400 pl Paraffin Dissolver per preparation up to approximately 15 mg ca 17 ul paraffin can be dissolved This cor responds 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 Larger amounts of paraffin can be dissolved by adding a higher volume of Paraffin Dissolver Cat No 740968 25 to the sample 30 ul per mg paraffin or by using xylene for deparaffinization as described in sections 5 3 and 5 4 When using more of the Paraffin Dissolver it is necessary to use a collection tube larger than 1 5 ml to enable removal of the lower aqueous phase after decrosslink step without spillage 2 5 Elution procedures High RNA and DNA concentrations in the elution fraction are desirable for all typical downstream applications In particular 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 nucleic acid if only small samples are processed Such RNA and DNA samples may even require a subsequent concentration to be suitable for the d
2. 20 C is recommended DISTRIBUTION AND USE IN THE USAIS PROHIBITED FOR PATENT REASONS 6 MACHEREY NAGEL 07 2010 Rev 02 Nucleic Acid Isolation from FFPE Samples 2 2 Kit specifications NucleoSpin FFPE RNA DNA is recommended for the isolation of total RNA and DNA from formalin fixed paraffin embedded FFPE tissue samples typi cally as thin sections approx 3 20 um thickness Thin sections mounted on glass slides can serve as sample material after transfer in a microcentrifuge tube Sample amount The maximum sample size is determined by a the amount of tissue and b by the amount of paraffin NucleoSpin FFPE RNA DNA 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 xylen RNA and DNA yield strongly depend on sample type quality amount and time of storage Further measured yield may vary considerably among differ ent quantification methods Yield determined by absorption measurement at 260 nm or via a fluorescent dye e g RiboGreen may deviate from values obtained by quantification via RT PCR Even quantification values obtained via RT PCR with a short e g 80 bp and a long e g 300 bp amplicon may differ considerably The deviation of
3. 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 analy sis and remaining paraffin tissue blocks are usually archived Existing extensive ar chives 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 RNA DNA analysis is limited due to chemical modification by formaldehyde and fragmentation of particularly the RNA during tissue processing sampling fixing em bedding and storage state time temperature of the samples Standard RNA isola tion procedures result in poor RNA yield or poor performance in downstream applica tions e g RT PCR A special purification system taking the unique requirements into account is inevitably necessary for successful analysis of nucleic acids from FFPE samples 2 1 The basic principle The NucleoSpin FFPE kits provide a convenient reliable and fast method to isolate RNA NucleoSpin FFPE RNA or RNA and DNA NucleoSpin FFPE RNA 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 depa raffinization Further the procedure omits the difficult removal of organic solvent from often barely visible
4. spin down briefly to clear the lid approx 1 s at 1 000 x g 100 pl FL 10 pl Proteinase K Vortex 60 C 15 min 3 hours 100 pl D Link Vortex 90 C 15 min 32 MACHEREY NAGEL 07 2010 Rev 02 RNA Isolation Xylene Deparaffinization 4 Adjust binding conditions 200 pl Add 200 pl ethanol 96 100 to the lysate and mix by ethanol vortexing 2 x 5 s Spin down briefly to clear the lid approx 1s at ones 1 000 x g 5 Bind RNA For each preparation take one NucleoSpin FFPE Column light blue ring placed in a Collection Tube Pipette lysate up and down two times before loading the Load lysate lysate Load the lysate into the column Centrifuge for 30 s at 2 000 x g 2 000 x g 30s The recommended centrifugation at 2 000xg 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 6a Desalt silica membrane Add 100 pi MDB Membrane Desalting Buffer and centrifuge at 11 000 x g for 30 s 100 pl MDB It is not necessary to use a fresh Collection Tube after this centrifugation step Salt removal will make the following rDNase digest much 11 000 X0 more effective If the column outlet has come into contact 30 s with the flow through for any reason discard the flow through and centrifuge again for 30 s at 11 000 xg MACHEREY NAGEL 07 2010 Rev 02 33 RNA Isolation Xylene De
5. 07 2010 Rev 02 RNA and DNA Isolation Xylene Deparaffinization Lyse sample Add 100 pl Buffer FL and 10 pl 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 flushed 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 sam ple tissue If a large portion of sample is still undigested add additional 10 ul Proteinase K solution and continue digestion for fur ther 3 hours An overnight incubation is only recommended if the tissue is not well digested within 2 x 3 hours Release of DNA generally requires longer digestion times than release of RNA Note During this incubation step protein is digested and nucleic acids are released into solution Vortex tube 5 s 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 pl Decrosslink Buffer D Link to the lysate and vortex vigorously 5 s Incubate at 90 C for exactly 15 min Subsequently vortex 5 s and cool down to room temper ature for approx 2 min If necessary spin down briefly t
6. DNA purification from FFPE samples with xylene deparaffinization RNA purification from FFPE samples with xylene deparaffinization Support protocol NucleoSpin FFPE RNA DNA DNA digestion in the RNA eluate 6 Appendix 6 1 6 2 6 3 6 4 6 5 Comments on RNA quality and quantity Troubleshooting Ordering information References Product use restriction warranty ana A A z at i _ O O O O NOQA 15 15 21 26 31 36 37 37 38 42 43 43 MACHEREY NAGEL 07 2010 Rev 02 Nucleic Acid Isolation from FFPE Samples 1 Components 1 1 Kit contents NucleoSpin FFPE RNA DNA 10 preps 50 preps 250 preps Cat No 740978 10 740978 50 740978 250 Paraffin Dissolver 5 ml 25 ml 125 ml Lysis Buffer FL 1 8 ml 8 ml 30 ml Decrosslink Buffer D Link 1 8 ml 8 ml 30 ml Wash Buffer FW1 2x1 mi 15 ml 2x15 mi H ewran as 2 mi 7 mi 2x 20 ml a Desalting Buffer 1 8 ml 10 ml 50 ml Reaction Buffer for rDNase 0 5 ml 3 ml 20 ml rDNase RNase free 1 vial 1 vial 2 vials lyophilized size A size C size D Proteinase K lyophilized 6 mg 30 mg 75 mg Proteinase Buffer PB 0 8 ml 1 8 ml 8 ml DNA Wash Concentrate 2 ml 4 ml 12 ml DNA Elute 1 2 ml 6 ml 3x6 ml RNase free H O 5 ml 15 ml 25 ml NucleoSpin FFPE Columns light blue rings 10 50 250 plus Collection Tubes Collection Tubes 2 ml 20 2x50 2 x 250 Collection Tubes 1 5 ml 10 50 250 User Manual 1 1 1 For preparation of working solutions and stor
7. Elute solution does not inhibit microbial growth Be aware not to contaminate DNA Elute with any source of microbial contamination DNA is commonly stable even at 37 C for 2h with or without addition of a typical restriction enzyme buffer Thus eluted DNA is free of any measurable DNase activity Store eluted DNA for short term at 0 4 C and freeze at 20 C for long term storage 10 MACHEREY NAGEL 07 2010 Rev 02 Nucleic Acid Isolation from FFPE Samples 3 Storage conditions and preparation of working solutions Attention Buffers FW1 FL and MDB contain chaotropic salts Wear gloves and goggles 96 100 ethanol is required to adjust the binding conditions in the lysate Storage conditions Store lyophilized rDNase and Proteinase K at 4 C on arrival stable up to 1 year All other kit components should be stored at room temperature 18 25 C and are stable up to one year Storage at lower temperatures may cause precipita tion of salts Before starting any NucleoSpin FFPE protocol prepare the following rDNase Add indicated volume see next page or on the vial of RNase free water to the rDNase vial and incubate for 1 min at room temperature Gently swirl the vial to completely dissolve the rDNase Be careful not to mix rDNase vigorously as rDNase is sensitive to mechanical agitation Dispense into aliquots and store at 20 C The frozen working solution is stable for 6 months Do not freeze thaw th
8. Incubate at room temperature for 15 min 15 min It is not necessary to use a new Collection Tube after the incubation step MACHEREY NAGEL 07 2010 Rev 02 19 RNA and DNA Isolation Paraffin Dissolver 7 Wash and dry silica membrane 1 wash 100 pl FW1 Add 100 jil Buffer FW1 to the NucleoSpin FFPE RT Column Incubate for 2 min at RT a 2 min Centrifuge for 30 s at 11 000 x g 11 000 x g Discard Collection Tube with flow through and place the 30 s column into a new Collection Tube 2 ml Buffer FW1 will inactivate the rDNase Add 400 jil Buffer FW2 to the NucleoSpin FEPE J apo jil FW2 Column Centrifuge for 30 s at 11 000 x g ae g Discard flow through and place the column back into the e gt Collection Tube Add 200 HI Buffer FW2 to the NucleoSpin FFPE Column Centrifuge for 2 min at 11 000 x g to dry the membrane 200 pl FW2 Discard the Collection Tube with flow through and place 11 000 x g the column into a nuclease free 1 5 ml Collection Tube 2 min provided If for any reason the liquid level in the Collection Tube reaches the NucleoSpin FFPE Column after centrifugation discard flow through and centrifuge again 8 Elute highly pure RNA 10 pl H O Elute the RNA in 10 pl H O RNase free supplied and RNase free centrifuge at 11 000 x g for 30 s If higher RNA concentration or higher elution volume is desired elution volume may be varied in the range of eS 11 000 x g 5 30 ul 30s 20 MACHEREY
9. Paraffin Dissolver 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 Pipette 10 pl 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 exces sively 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 L of the pre mix 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 60 C for 15 min 3 hours to lyse sample tissue If residual unlysed tissue particles are visible after 15 min incubation continue the incubation for up to 3 hours If a large portion of sample is still undigested add additional 10 ul Proteinase K solution and continue digestion for further 3 hours An overnight incubation is only recommended if the tissue is not well digested within 2x 3 hours RNA is generally sensitive to autolysis Thus a short inclubation time is prefer able as long as th
10. nucleic acids generally show better performance in downstream applications MACHEREY NAGEL 07 2010 Rev 02 17 RNA and DNA Isolation Paraffin Dissolver 4 Adjust binding condition 200 ul Add 200 ul ethanol 96 100 to the tube and mix by ethanol vortexing 2 x 5 s Vori ortex Centrifuge for 30 s at 11 000 x g to achieve complete phase separation ed 11 000 x g 30s The ethanol will merge with the aqueous lower phase only 5 Bind RNA and DNA For each preparation take one NucleoSpin FFPE Column light blue ring placed in a Collection Tube Pipette aqueous lower phase completely into the NucleoSpin FFPE Column oe oa It is recommended to pipette a volume of 450 ul on the spin aqueous column to ensure that the complete aqueous lower phase 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 e gt 2 000 x g 30 s Centrifuge for 30 s at 2 000 x g The recommended centrifugation at 2 000xg 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 6a Wash and dry silica membrane 1 wash weme DNA Wash Add 100 pl DNA Wash and centrifuge at 11 000 x g for 30 s It is not necessary to use a fresh Collection Tube 11 000 x g after this centrifugation step 30s Note MDB is not used if RN
11. or operation MACHEREY NAGEL 07 2010 Rev 02 45
12. pH influence A absorption as well as ratio Asd A20 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 measure ment of purity of nucleic acids Biotechniques 19 208 209 Wilfinger W W Mackey K and Chomczyski P 1997 Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity Biotechniques 22 474 481 Proteinase digestion time Depending of the nature of the sample an optimal digestion time from 15 min to 2x 3 hours has to be determined empiri cally If tissue residues are still visible after 15 min continue the incubation for up to 3 hours If still a major amount of sample is undigested continue digestion overnight An overnight incuba tion is not recommended if the tissue digested well within 2x 3 hours Sample material Too much starting material used Overloading may lead to de creased overall yield Reduce amount of sample material or use larger volume of Buffer FL Insufficient disruption and or homogenization of starting materi al Make sure that the lysate is clear before loading the lysate on the column If the column is clogged try spinning at full speed for two minutes in order to save the sample MACHEREY NAGEL 07 2010 Rev 02 39 Nucleic Acid Isolation from FFPE Samples rDNase not active e Reconstitute and store lyophilized rDNase according to instruc tions gi
13. 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 di gestion for further 3 hours An overnight incubation is only RT recommended if the tissue is not well digested within 2 x 3 3 hours hours Release of DNA generally requires longer digestion times than release of RNA Note During this incubation step protein is digested and nucleic acids are released into solution Vortex 5 s Vortex 5s 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 100 pl D Link Add 100 pl Decrosslink Buffer D Link to the tube and vortex gently to mix Buffer D Link into the aqueous lower Vortex phase F 11 000 x Centrifuge at 11 000 x g for 30 s to obtain phase forma cs 30 s g tion Incubate at 90 C for exactly 15 min Vortex 5 s and let cool down to room temperature approx 2 min 90 C If necessary spin down briefly to clear the lid approx 1 s at 15 min 1 000 x 9 Vortex Note This decrosslink step is necessary to remove the crosslinks chemical modifications caused by formalin from the nucleic acids which were released into solution by the previous lysis step Decrosslinked
14. 0 s 6a _ Desalt silica 100 pl MDB 100 pl MDB membran Su ek E e 11 000 x g 30s 11 000 x g 30s 6b Digest DNA 25 ul rDNase mixture 25 ul rDNase mixture RT 15 min RT 15 min 7 Wash and dry silica 4s 100 pl FW1 100 pl FW1 membrane RT 2 min RT 2 min a 11 000 x g 30s 11 000 x g 30s ou 400 pl FW2 400 pl FW2 yg S 11 000 x g 30s 11 000 x g 30s 3 200 pl FW2 200 pl FW2 eS 11 000 x g 2 min 11 000 x g 2 min 8 Elute highly pure 10 pl RNase free H O 10 pl RNase free H O RNA m g5 11 000 x g 30 s 11 000 x g 30 s MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 D 52355 D ren Germany Tel 49 0 24 21 969 270 www mn net com e mail tech bio mn net com Nucleic Acid Isolation from FFPE Samples Table of contents 1 Components 1 1 1 2 1 3 Kit contents Reagents consumables and equipment to be supplied by user About this User Manual 2 Product description 2 1 2 2 2 3 2 4 2 5 2 6 2 7 The basic principle Kit specifications Handling preparation and storage of starting materials Quantities of FFPE sections Elution procedures Stability of isolated RNA Stability of isolated DNA 3 Storage conditions and preparation of working solutions 4 Safety instructions risk and safety phrases 5 Protocols 5 1 5 2 5 3 5 4 5 5 RNA and DNA purification from FFPE samples using Paraffin Dissolver RNA purification from FFPE samples using Paraffin Dissolver RNA and
15. A and DNA are isolated 2 wash 100 ul i DNA Wash Add 100 pl DNA Wash and centrifuge at 11 000 x g for 2 min to dry the membrane 11 000 x g Discard Collection Tube with flow through 2 min 18 MACHEREY NAGEL 07 2010 Rev 02 RNA and DNA Isolation Paraffin Dissolver 6b Elute DNA Place the NucleoSpin FFPE Column into a 1 5 ml microcentrifugation tube not supplied and apply 20 pl DNA Elute directly onto the center of the silica mem brane of the column Incubate for 2 min at RT 20 pl DNA Elute Elute the DNA by centrifugation for 30 s at 11 000 x g RT If higher DNA concentrations or higher elution volumes 2 min are desired elution volume may be carefully varied in the range of 10 30 ul see section 2 5 11 000 x g Caution The temperature of DNA Elute solution should not ae exceed 30 C otherwise RNA will be partly eluted as well Eluted DNA is immediately ready for downstream applica tions without further purification Place NucleoSpin FFPE Column into a new Collection Tube to continue with RNA isolation 6c Digest residual DNA Prepare rDNase reaction mixture in a sterile microcen 25 ul trifuge tube not provided For each isolation add 3 pl DN H reconstituted rDNase see section 3 to 27 pl Reaction r ase Buffer for rDNase Mix by flicking the tube reaction mixture Apply 25 pl rDNase reaction mixture directly onto the center of the silica membrane of the column Close the RT lid
16. EL 07 2010 Rev 02 25 RNA and DNA Isolation Xylene Deparaffinization 5 3 RNA and DNA purification from FFPE samples with xylene deparaffinization Before starting the preparation Check if rDNase Proteinase K DNA Wash and Buffer FW2 were prepared according to section 3 Check if 96 100 ethanol is available Set incubator s at 60 C for ethanol evaporation and lysis step 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 Deparaffinize sample Add 1 ml xylene or alternatives see section 1 2 to the sample Incubate at room temperature until the paraffin is completely dissolved usually approx 2 min and vortex vigorously 10 s Centrifuge for 2 min at full speed Discard the supernatant by pipetting Do not remove any of the pellet Add 1 ml ethanol 96 100 to the pellet and vortex 5s Centrifuge for 2 min at full speed Discard the supernatant by pipetting Do not remove any of the pellet 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 RNA and DNA yield v 1 ml xylene RT 2 min Vortex full speed 2 min Discard supernatant 1 ml ethanol Vortex full speed 2 min Discard supernatant 60 C 3 10 min 26 MACHEREY NAGEL
17. NAGEL 07 2010 Rev 02 RNA Isolation Paraffin Dissolver 5 2 RNA purification from FFPE samples using Paraffin Dissolver Before starting the preparation Check if rDNase Proteinase K and Buffer FW2 were prepared according to section 3 Check if 96 100 ethanol is available Set incubator s at 60 C for paraffin melting and lysis step and 90 C for de crosslink step Sample preparation Insert FFPE section s in a microcentrifuge tube not sup plied For appropriate sample amounts see section 2 4 Deparaffinize sample Add 400 ul Paraffin Dissolver to the sample 400 ul Paraffin Incubate 3 min at 60 C to melt the paraffin Dissolver Vortex the sample immediately at 60 C at a vigorous 60 C speed to dissolve the paraffin 3 min Cool down sample to room temperature Vortex Make sure that paraffin completely melts during the heat incu hot sample bation step and mix well after melting to completely dissolve the paraffin Insufficient mixing of the heated sample may cause recur rence 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 For samples comprising more than 15 mg paraffin use 30 ul Paraffin Dissolver per 1 mg paraffin If more than 400 ul Paraffin Dissolver is necessary place sample in a 2 ml tube not provided MACHEREY NAGEL 07 2010 Rev 02 21 RNA Isolation
18. RNA and DNA Isolation from FFPE Samples User Manual NucleoSpin FFPE RNA DNA July 2010 Rev 02 MACHEREY NAGEL MN RNA and DNA Isolation from FFPE Samples Protocol at a glance Rev 02 NucleoSpin FFPE RNA DNA Protocol 5 3 RNA and DNA Protocol 5 1 RNA and DNA isolation with Paraffin Dissolver isolation with xylene 1 Deparaffinize sample 400 ul Paraffin Dissolver 1 ml xylene 60 C 3 min RT 2 min Mix hot sample Mix Full speed 2 min Letsamplecooldown Discard supernatant 1 ml 98 ethanol Mix Full speed 2 min Discard supernatant Dry at 60 C 3 10 min 2 Lyse sample 100 pl FL 100 pl FL 11 000 x g 1 min j 10 pl Proteinase K 10 pl Proteinase K Mix Mix RT 3 hours RT 3 hours 3 Decrosslink 100 pl D Link 100 pl D Link Mix gently Mix gently 5 11 000 x g 30s S 90 C 15 min 90 C 15 min 4 Adjust binding 200 pl 98 ethanol 200 ul 98 ethanol condition f Mix Mix eS 11 000 x g 30s 5 Bind RNA DNA E Load aqueous lower phase Load lysate J 2 000 x g 30 s 2 000 x g 30 s 6a Wash and dry silica i 100 pl DNA Wash 100 pl DNA Wash membrane z e 11 000 x g 30s 11 000 x g 30s 8 2na 100 pl DNA Wash 100 pl DNA Wash Ss 11 000 x g 2 min 11 000 x g 2 min 6b Elute DNA 20 ul DNA Elute 20 pl DNA Elute E RT 2 min RT 2 min eS 11 000 x g 30s 11 000 x g 30s 6c Digest residual DNA 25 ul DNase mixture 25 ul DNase mixture RT 15 min RT 15 min 7 Wa
19. SS 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 spe cial including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with 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 state ments 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 i
20. age conditions see section 3 4 MACHEREY NAGEL 07 2010 Rev 02 Nucleic Acid Isolation from FFPE Samples 1 2 Reagents consumables and equipment to be supplied by user Reagents e 96 100 ethanol to adjust binding conditions and to prepare Wash Buffer FW2 undenaturated is preferable e 50 ethanol to prepare DNA Wash undenaturated is preferable e Optional for deparaffinization without Paraffin Dissolver Xylene or d Limonene e g Roti Histol Hemo De or mixtures of isoparafinic hydrocarbons e g Roticlear Micro Clear Neo Clear Consumables e 1 5 ml microcentrifuge tubes for sample lysis and DNA elution e Disposable pipette tips Equipment Manual pipettors e Centrifuge for microcentrifuge tubes e Vortex mixer e Thermal heating block adjustable to 60 C and 90 C e 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 FFPE RNA DNA 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 glance is designed to be used only as a supplemental tool for quick referencing while performing the purifica tion procedure All technical literature is available on the internet at www mn net com MACHEREY NAGEL 07 2010 Rev 02 5 Nucleic Acid Isolation from FFPE Samples 2 Product description
21. ation Xylene Deparaffinization Lyse sample Add 100 pl Buffer FL and 10 pl 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 flushed back into the solution by pipetting Pipette solution up and down in order to homogenize sections Incubate at 60 C for 15 min 3 hours to lyse sample tissue If residual unlysed tissue particles are visible after 15 min incubation continue the incubation for up to 3 hours If a large portion of sample is still undigested add additional 10 ul Proteinase K solution and continue digestion for further 3 hours An overnight incubation is only recommended if the tissue is not well digested within 2 x 3 hours RNA is generally sensitive to autolysis Thus a short incubation time is preferable as long as the tissue is lysed sufficiently Vortex tube 5 s 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 lysate and vortex vigorously 5 s Incubate at 90 C for exactly 15 min Subsequently vortex 5s and cool down to room temperature for approx 2 min If necessary
22. d A seo Ago ratio Measurement not in the range of photometer detection limit In order to obtain a reliable A A ratio it is necessary that the initially measured A and A values are significantly above the detection limit of the photometer used An A value close to the background noise of the photometer will cause non reliable A eo ratios MACHEREY NAGEL 07 2010 Rev 02 41 Nucleic Acid Isolation from FFPE Samples 6 3 Ordering information Product Cat No Pack of NucleoSpin FFPE RNA DNA 740978 10 50 250 10 50 250 NucleoSpin FFPE RNA 740969 10 50 250 10 50 250 NucleoSpin RNA XS 740902 10 50 250 10 50 250 NucleoSpin RNA Clean up XS 740903 10 50 250 10 50 250 NucleoSpin RNA II ee 10 20 50 250 NucleoSpin RNA L 740962 20 20 NucleoSpin RNA Protein 740933 10 50 250 10 50 250 NucleoSpin TriPrep 740966 10 50 250 10 50 250 NucleoSpin RNA DNA Buffer Set rDNase Set Paraffin Dissolver NucleoSpin Filters Collection Tubes 2 ml Visit www mn net com for more detailed product information 740944 740963 740968 25 740606 740600 Suitable for 100 preps 1 set 25 ml 50 1000 DISTRIBUTION AND USE IN THE USAIS PROHIBITED FOR PATENT REASONS 42 MACHEREY NAGEL 07 2010 Rev 02 Nucleic Acid Isolation from FFPE Samples 6 4 References Castiglione F et a 2007 Real time PCR analysis of RNA extracted from formalin fixed a
23. d flow through and centrifuge again 34 MACHEREY NAGEL 07 2010 Rev 02 RNA Isolation Xylene Deparaffinization Elute highly pure RNA 10 pI H O Elute the RNA in 10 pl H O RNase free supplied and he m kak centrifuge at 11 000 x g for 30 s If higher RNA concentration or higher elution volume is 11 000 x desired elution volume may be varied in the range of g 5 30 ul 30 s MACHEREY NAGEL 07 2010 Rev 02 35 NucleoSpin FFPE RNA DNA 5 5 Support protocol NucleoSpin FFPE RNA DNA DNA digestion in the RNA eluate Comments on DNA digestion Although the on column rDNase digestion in the standard protocol is very efficient there are still certain applications which require even lower contents of residual DNA The removal of DNA to a completely undetectable level is challenging and the efficiency of an on column DNA digestion is sometimes not sufficient for downstream applications requiring lowest residual content of DNA Atypical example for such a demanding application is an RT PCR reaction in which the primer molecules do not differentiate between cDNA derived from RNA and contami nating genomic DNA Especially if e high copy number targets are analyzed e g multi gene family mitochondrial plastidal or plasmid targets from transfections e the target gene is of a very low expression level e the amplicon is relatively small lt 200 bp DNA digestion in solution can efficiently deg
24. disposable polypropylene tubes is recommended Keep tubes closed whenever possible during the preparation Glassware should be oven baked for at least 2 hours at 250 C before use Poor sample quality Sample quality very much influences the obtainable RNA amount and quality For aspects concering sample harvest fixation embeding and storage refer to Castiglione et al 2007 Chung et al 2008 Leyland Jones et al 2008 von Ahlfsen et al 2007 von Maldegem et al 2008 Reagents not applied or restored properly Reagents not properly restored Add the indicated volume of RNase free H O to rDNase vial and 96 ethanol to Buffer FW2 Concentrate and mix Reconstitute and store lyophilized rDNase and 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 lysis Binding of RNA to the silica membrane is only effective in the presence of ethanol 38 MACHEREY NAGEL 07 2010 Rev 02 Nucleic Acid Isolation from FFPE Samples Poor RNA quality or yield continued Clogged NucleoSpin Column Poor RNA quality or yield Kit storage Reconstitute and store lyophilized rDNase according to instruc tions given in section 3 Store kit components as described in section 3 Keep bottles tightly closed in order to prevent evaporation or contamination lonic strength and
25. e aliquots more than three times Proteinase K Add the indicated volume see next page or on the vial of Proteinase Buffer PB to dissolve lyophilized Proteinase K Proteinase K solu tion is stable at 20 C for 6 months DNA Wash Add indicated volume see next page or on the bottle of 50 ethanol to the DNA Wash Concentrate Store Buffer DNA Wash at room tem perature 18 25 C for up to one year Wash Buffer FW2 Add the indicated volume see next page or on the bottle of 96 100 ethanol to Buffer FW2 Concentrate Store Buffer FW2 at room temperature 18 25 C for up to one year MACHEREY NAGEL 07 2010 Rev 02 11 Nucleic Acid Isolation from FFPE Samples NucleoSpin FFPE RNA DNA 10 preps 50 preps 250 preps Cat No 740978 10 740978 50 740978 250 Wash 2 ml 7 ml 2x20 ml Buffer FW2 Add 8 ml Add 28 ml Add 80 ml Concentrate 96 100 ethanol 96 100 ethanol 96 100 ethanol to each bottle DNA Wash 2ml 4ml 12 ml Concentrate Add 8 ml Add 16 ml Add 48 ml 50 ethanol 50 ethanol 50 ethanol rDNase 1 vial size A 1 vial size C 2 vials size D RNase free lyophilized Add 55 ul Add 230 ul Add 540 ul RNase free H O RNase free H O RNase free H O to each vial 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 07 2010 Rev 02 Nucleic Acid Isolation from FFPE Samples 4 Safety instruc
26. e silica membrane of the column Close the RT lid Incubate at room temperature for 15 min 15 min It is not necessary to use a new Collection Tube after the incubation step MACHEREY NAGEL 07 2010 Rev 02 29 RNA and DNA Isolation Xylene Deparaffinization 7 Wash and dry silica membrane 1 wash 100 pl FW1 Add 100 l Buffer FW1 to the NucleoSpin FFPE RT Column Incubate for 2 min at RT 2 min Centrifuge for 30 s at 11 000 x g 11 000 x g Discard Collection Tube with flow through and place the 30s column into a new Collection Tube 2 ml Buffer FW1 will inactivate the rDNase Add 400 pl BufferFW2 to the NucleoSpin FFPE 4 400 jil FW2 Column i Centrifuge for 30 s at 11 000 x g e g Discard flow through and place the column back into the C Collection Tube Add 200 HI Buffer FW2 to the NucleoSpin FFPE Column Centrifuge for 2 min at 11 000 x g to dry the membrane 200 pl FW2 Discard the Collection Tube with flow through and place 11 000 x g the column into a nuclease free 1 5 ml Collection Tube 2 min provided If for any reason the liquid level in the Collection Tube reaches the NucleoSpin FFPE Column after centrifugation discard flow through and centrifuge again 8 Elute highly pure RNA 10 pl H O Elute the RNA in 10 pl H O RNase free supplied and RNase free centrifuge at 11 000 x g for 30s If higher RNA concentration or higher elution volume is desired elution volume may be varied in t
27. e tissue is lysed sufficiently Vortex 5 s 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 100 pl FL Vortex 11 000 x g 1 min 10 jil Proteinase K Mix by pipetting up and down 60 C 3 hours Vortex 5s 22 MACHEREY NAGEL 07 2010 Rev 02 RNA Isolation Paraffin Dissolver Decrosslink Add 100 pl Decrosslink Buffer D Link to the tube and vortex gently to mix Buffer D Link into the aqueous lower phase Centrifuge at 11 000 x g for 30s to obtain phase formation Incubate at 90 C for exactly 15 min Vortex 5 s 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 Adjust binding conditions Add 200 pl ethanol 96 100 to the tube and mix by vortexing 2 x 5 s Centrifuge for 30s at 11 000 x g to achieve complete phase separation The ethanol will merge with the aqueous lower phase only Bind RNA For each preparation take one NucleoSpin FFPE Column light blue ring placed in a Collection Tube Pipette aqueous lower phase completely into the NucleoSpin FFPE Column It is recommended to pipette a volume of 450 ul on the spin column to ensure that the complete aqueous lower phase is transferred the volume of the aqueous phase is approx 410 ul Sma
28. epending on the robustness of the used RT PCR system RT PCR might be inhibited if complete eluates are used as template for RT PCR Use less eluate as template 40 MACHEREY NAGEL 07 2010 Rev 02 Nucleic Acid Isolation from FFPE Samples Suboptimal performance of RNA in downstream experiments continued Store isolated RNA properly Eluted RNA should always be kept on ice for optimal stability since trace contaminations of omnipresent RNases general lab ware fingerprints dust will degrade the isolated RNA For short term storage freeze at 20 C for long term storage freeze at 70 C Discrepancy between A quantifica tion values and PCR quantifica tion values Silica abrasion from the membrane Due to the typically low RNA content in very small samples and the resulting low total amount of isolated RNA a RNA quan tification via A s 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 o quantification of small RNA amounts centrifuge the eluate for 30s at gt 11 000 x g and take an aliquot for measurement without disturbing any sediment Alternatively use a silica abra sion insensitive RNA quantification method e g RiboGreen fluorescent dye Unexpecte
29. er included in the kit and one utilizes xylene or comparable organic solvents not supplied with the kit The table below gives an overview of the protocols for RNA isolation and RNA and DNA isolation Deparaffinization with Paraffin Dissolver is recommended for highest convenience Table 2 Overview of protocols RNA and DNA isolation RNA isolation Deparaffinization with Section 5 1 Section 5 2 Paraffin Dissolver Deparaffinization with xylene Section 5 3 Section 5 4 5 1 RNA and DNA purification from FFPE samples using Paraffin Dissolver Before starting the preparation Check if rDNase Proteinase K DNA Wash and Buffer FW2 were prepared according to section 3 e Check if 96 100 undenatured ethanol is available e Set incubator s at 60 C for paraffin melting and 90 C for decrosslink step Sample preparation Provide FFPE section s in a microcentrifuge tube not supplied For appropriate sample amounts see section 2 4 MACHEREY NAGEL 07 2010 Rev 02 15 RNA and DNA Isolation Paraffin Dissolver 1 Deparaffinize sample Add 400 ul Paraffin Dissolver to the sample Incubate 3 min at 60 C to melt the paraffin Vortex the sample immediately at 60 C at a vigorous 400 ul speed to dissolve the paraffin k lana Paraffin Cool down sample to room temperature Dissolver Make sure that paraffin completely melts during the heat incu 60 C bation step and mix well after melting to c
30. esired application NucleoSpin FFPE kits allow efficient elution in small volume Elution volumes in the range of 5 30 pl are recommended for RNA the default volume is 10 ul During DNA elution RNA stays bound to the column The DNA washing solution DNA Wash and the DNA elution buffer DNA Elute are finely tuned to achieve this Therefore the default DNA elution volume of 20 ul should be kept or altered only in the range of 10 30 ul The temperature of the DNA Elute solution should not exceed 30 C otherwise RNA will partly elute with the DNA Elute solution DNA Elute solution may stay for 1 5 min on the column for elution before spinning down Eluted DNA is immediately ready for downstream applications without further purification MACHEREY NAGEL 07 2010 Rev 02 9 Nucleic Acid Isolation from FFPE Samples 2 6 Stability of isolated RNA Eluted RNA should immediately be put on ice and always kept on ice during work for optimal stability Contamination with almost omnipresent RNases general lab ware fingerprints dust may be a risk for isolated RNA For short term storage freeze at 20 C for long term storage freeze at 70 C 2 7 Stability of isolated DNA Due to its composition the DNA elution buffer DNA Elute does not inhibit DNases DNA Elute does not contain substances e g EDTA to complex divalent cations Therefore be aware not to contaminate DNA Elute with DNases Further due to its composition DNA
31. g for 2 min to dry the membrane Discard Collection Tube with flow through 200 ul ethanol Vortex Load lysate 2 000 x g 30s 100 pl DNA Wash 11 000 x g 30s 100 pl DNA Wash 11 000 x g 2 min 28 MACHEREY NAGEL 07 2010 Rev 02 RNA and DNA Isolation Xylene Deparaffinization 6b Elute DNA Place the NucleoSpin FFPE Column into a 1 5 ml microcentrifugation tube not supplied and apply 20 ul DNA Elute directly onto the center of the silica membrane of the column Incubate for 2 min at RT 20 pl DNA Elute Elute the DNA by centrifugation for 30 s at 11 000 x g RT If higher DNA concentrations or higher elution volumes 2min are desired elution volume may be carefully varied in the range of 10 30 ul see section 2 5 11 000 x g Caution The temperature of DNA Elute solution should not aus exceed 30 C otherwise RNA will be partly eluted as well Eluted DNA is immediately ready for downstream applica tions without further purification Place NucleoSpin FFPE Column into a new Collection Tube to continue with RNA isolation 6c Digest residual DNA Prepare rDNase reaction mixture in a sterile microcen 25 ul trifuge tube not provided For each isolation add 3 pl DN H reconstituted rDNase see section 3 to 27 pl Reaction ease Buffer for rDNase Mix by flicking the tube reaction mixture Apply 25 pl rDNase reaction mixture directly onto the center of th
32. he range of eS 11 000 x g 5 30 ul 30s 30 MACHEREY NAGEL 07 2010 Rev 02 RNA Isolation Xylene Deparaffinization 5 4 RNA purification from FFPE samples with xylene depar affinization Before starting the preparation Check if rDNase Proteinase K and Buffer FW2 were prepared according to section 3 Check if 96 100 ethanol is available Set incubator s at 60 C for ethanol evaporation and lysis step 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 Deparaffinize sample 1 ml xylene Add 1 ml xylene or alternatives see section 1 2 to the sample a min Incubate at room temperature until the paraffin is com pletely dissolved usually approx 2 min and vortex vigor Vortex ously 10 s iilispead Centrifuge for 2 min at full speed 2 min Discard Discard the supernatant by pipetting Do not remove supernatant any of the pellet Add 1 ml ethanol 96 100 to the pellet and vortex 1 ml ethanol 5s Vortex Centrifuge for 2 min at full speed Discard the supernatant by pipetting Do not remove full speed any of the pellet Bi ae iscar supernatant Incubate the open tube at 60 C for 3 10 min to dry the pellet 60 C It is important to evaporate all residual ethanol Residual 3 10 min ethanol may reduce RNA yield MACHEREY NAGEL 07 2010 Rev 02 31 RNA Isol
33. ill show a high RIN RNA Integrity Number Agilent or RQI RNA Quality Indicator BioRad value e A low RNA integrity i e highly degraded RNA with fragment sizes exclusive ly below 200 nucleotides will certainly not enable amplification of fragments exceeding this size However it is still likely that small sized target sequences can be amplified successfully especially if they are well decrosslinked Neither RNA yield nor RIN RQI absorbance ratios or size distribution can reliably predict the performance in downstream RT PCR applications especially if different purification and decrosslinking systems are compared The major quality indicator for RNA isolated from FFPE samples is its performance in RNA profiling analysis i e in RT PCR or microarray experiments MACHEREY NAGEL 07 2010 Rev 02 37 Nucleic Acid Isolation from FFPE Samples 6 2 Troubleshooting Problem Possible cause and suggestions Incomplete paraffin dissolution in xylene Hard to solve paraffin Incubate sample 5 min in xylene at 60 C and mix Centrifuge 2 min at maximum speed and remove supernatant If paraffin is still not dissolved completely add again 1 ml xylene incubate 2 min at 60 C centrifuge at maximum speed and discard su pernatant Poor RNA quality or yield RNase contamination Create an RNase free working environment Wear gloves dur ing all steps of the procedure Change gloves frequently Use of sterile
34. l assays such as gel electrophoresis spectrophotometry fluorome try or microfluidics analysis However efficiency of enzymatic reactions with chemically modified RNA is significantly decreased Affected RNA analysis methods and applications are for example e Spectrophotometry e g absorption measurement A A gt Asa e Fluorometry e g RiboGreen e Denaturing agarose gel electrophoresis e Mirofluidics analysis e g Agilent 2100 Bioanalyzer BioRad s Experion Automated Electrophoresis System e RT PCR e Array analysis e g DNA microarrays The following aspects should be considered when applying one of the listed methods especially when comparing efficiency of different RNA isolation procedures and usability of the isolated RNA e A high RNA yield as determined by A readings or by fluorescent dye e g RiboGreen analysis does not necessarily result in good performance of the RNA in an RT PCR RNA may be highly degraded i e smaller than the RT PCR target or insufficiently decrosslinked e Low or no RNA yield as determined by A readings will most likely result in poor RT PCR results but it is still possible to achieve a good performance There may be a small amount RNA which is decrosslinked sufficiently and shows good reactivity A high RNA integrity does not guarantee a good amplifiability of RNA in RT PCR or reactivity in other enzymatic reactions RNA may be insufficiently decrosslinked but st
35. ll carry over of the organic upper phase has no negative effect on the binding procedure Centrifuge for 30 s at 2 000 x g The recommended centrifugation at 2 000 x g is more effi cient than centrifugation at 11 000 x g Discard Collection Tube with flow through and place the column in a new Collection Tube 2 ml T v 100 pl D Link Vortex 11 000 x g 30 s 90 C 15 min Vortex 200 ul ethanol Vortex 11 000 x g 30s Load aqueous lower phase 2 000 x g 30s MACHEREY NAGEL 07 2010 Rev 02 23 RNA Isolation Paraffin Dissolver 6a Desalt silica membrane Add 100 ui MDB Membrane Desalting Buffer and centrifuge at 11 000 x g for 30 s 100 pl MDB It is not necessary to use a fresh Collection Tube after this centrifugation step 11 000 x g Salt removal will make the following rDNase digest much 30s more effective If the column outlet has come into contact with the flow through for any reason discard the flow through and centrifuge again for 30 s at 11 000 x g 6b Digest DNA Prepare rDNase reaction mixture in a sterile microcen 25 ul trifuge tube not provided For each isolation add 3 j l DN H reconstituted rDNase see section 3 to 27 pl Reaction r ase Buffer for rDNase Mix by flicking the tube reaction mixture Apply 25 pl rDNase reaction mixture directly onto the center of the silica membrane of the column Close the RT lid Incubate at
36. nd paraffin embeded tissues effects of the fixation on outcome reliability Appl Immunohistocehm Mol Morphol 15 3 338 342 Chung J Y et al 2008 Factors in tissue handling and processing that impact RNA obtained from formalin fixed paraffin embedded tissue Journal of Histochemistry amp Cytochemistry 56 11 1033 1042 Koch et al 2006 Real time quantitative RT PCR shows variable assay dependent sensitivity to formalin fixation implications for direct comparison of transcript levels in paraffin embedded tissues Diagn Mol Pathol 15 3 149 156 Leyland Jones B R et al 2008 Recommendations for collection and handling of specimens from group breast cancer clinical trials J Clin Oncol 26 34 5638 5644 Penland S K e al 2007 RNA expression analysis of formalin fixed paraffin embed ded tumors Laboratory investigation 87 383 391 von Ahlfen S et al 2007 Determinants for RNA quality from FFPE samples PLoS ONE Issue 12 e1261 von Maldegem F etal 2008 Effects of processing delay formalin fixation and immunohistochemistry on RNA recovery from formalin fixed paraffin embedded tissue sections Diagn Mol Pathol 17 1 51 58 6 5 Product use restriction warranty NucleoSpin FFPE RNA DNA kit components were developed designed distributed and sold FOR RESEARCH PURPOSES ONLY They are suitable FOR IN VITRO USES ONLY No claim or representation is intended for its use to identify any specific orga
37. nformation 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 correctness of any of those applications Please contact MACHEREY NAGEL Germany Tel 49 0 24 21 969 270 e mail TECH BIO mn net com Last updated 12 2006 Rev 02 44 MACHEREY NAGEL 07 2010 Rev 02 Nucleic Acid Isolation from FFPE Samples Trademarks Micro Clear is a 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 RiboGreen is a registered trademark of Molecular Probes Inc Roticlear is a registered trademark of CARL ROTH GmbH amp Co KG Roti Histol is a registered trademark of CARL ROTH GmbH amp Co KG 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
38. nism or for clinical use diagnostic prognostic therapeutic or blood banking It is rather the responsibility of the user to verify the use of the NucleoSpin FFPE RNA DNA kit for a specific application range as the performance characteristic of this kit has not been verified to a specific organism This MACHEREY NAGEL product is shipped with documentation stating specifica 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 an extra copy MACHEREY NAGEL does not warrant against damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or im proper or abnormal use of this product against defects in products or components not MACHEREY NAGEL 07 2010 Rev 02 43 Nucleic Acid Isolation from FFPE Samples manufactured by MACHEREY NAGEL or against 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 EXPRE
39. o clear the lid approx 1 s at 1 000 x g 100 pl FL 10 pl Proteinase K Vortex RT 3 hours Vortex 5s 100 pl D Link Vortex 90 C 15 min MACHEREY NAGEL 07 2010 Rev 02 27 RNA and DNA Isolation Xylene Deparaffinization Note This decrosslink step is necessary to remove the crosslinks chemical modifications caused by formalin from the nucleic acids which were released into solution by the previous lysis step Decrosslinked nucleic acids generally show better performance in downstream applications Adjust binding conditions Add 200 pl ethanol 96 100 to the lysate and mix by vortexing 2 x 5 s Spin down briefly to clear the lid approx 1s at 1 000 x g 6a Bind RNA and DNA For each preparation take one NucleoSpin FFPE Column light blue ring placed in a Collection Tube 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 The recommended centrifugation at 2 000xg 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 100 pl DNA Wash and centrifuge at 11 000 x g for 30 s It is not necessary to use a fresh Collection Tube after this centrifugation step Note MDB is not used if RNA and DNA are isolated Add 100 pl DNA Wash and centrifuge at 11 000 x
40. ompletely dissolve 3 min the paraffin Insufficient mixing of the heated sample may cause recur Vortex rence of solid paraffin particles Make sure the sample does hot sample not comprise more than 15 mg paraffin or adjust the volume of Paraffin Dissolver see section 2 4 For samples comprising more than 15 mg paraffin use 30 ul Paraffin Dissolver per 1 mg paraffin If more than 400 pl Paraffin Dissolver is necessary place sample in a 2 ml tube not provided 2 Lyse sample Add 100 ul Buffer FL 100 pl FL Vortex vigorously Centrifuge at 11 000 x g for 1 min 11 000 x g Two phases will be formed a lower aqueous phase and an 1 min upper organic phase Tissue material will be transferred to the lower aqueous phase Pipette 10 pl Proteinase K solution directly into the lower aqueous phase 10 jil Proteinase K Mix the aqueous phase by pipetting up and down several times Pipette only the lower aqueous phase up and Mix by down Avoid mixing lower phase and upper phase exces pipetting up sively and down Make sure that the Proteinase K is mixed well with the lysis buffer 16 MACHEREY NAGEL 07 2010 Rev 02 RNA and DNA Isolation Paraffin Dissolver If multiple samples are processed preparation of a Buffer FL Proteinase K premix is recommended Add 110 ul of the pre mix to the reaction tube mix and centrifuge to achieve phase formation and to transfer the tissue into the aqueous lower phase Pipette aqueous phase
41. paraffinization 6b Digest DNA Prepare rDNase reaction mixture in a sterile microcen trifuge tube not provided For each isolation add 3 pl aes Hl reconstituted rDNase see section 3 to 27 pl Reaction r ase Buffer for rDNase Mix by flicking the tube reaction mixture Apply 25 pl rDNase reaction mixture directly onto the center of the silica membrane of the column Close the RT lid Incubate at room temperature for 15 min 15 min It is not necessary to use a new Collection Tube after the incubation step 7 Wash and dry silica membrane 1 wash 100 pl FW1 Add 100 pl BufferFW1 to the NucleoSpin FFPE RT Column Incubate for 2 min at RT 2 min Centrifuge for 30 s at 11 000 x g g g 11 000 x g Discard Collection Tube with flow through and place the 30s column into a new Collection Tube 2 ml Buffer FW1 will inactivate the rDNase Add 400 jll Buffer FW2 to the NucleoSpin FFPE 400 pl FW2 Column Centrifuge for 30 s at 11 000 x g Discard flow through 11 000 x g and place the column back into the Collection Tube 30 s Add 200ul Buffer FW2 to the NucleoSpin FFPE Column Centrifuge for 2 min at 11 000 x g to dry the membrane 200 pl FW2 Discard the Collection Tube with flow through and place 11 000 x g the column into a nuclease free 1 5 ml Collection Tube eS 2 min provided If for any reason the liquid level in the Collection Tube reaches the NucleoSpin FFPE Column after centrifugation discar
42. quantification also depends on nucleic acid size distribution as well as on efficiency of decrosslinking or extent of remaining crosslinks Please also see section 6 1 for considerations on determining RNA quality and quantity The innovative column design with a funnel shaped thrust ring and a small silica membrane area allows elution of RNA and DNA in as little as 5 30 ul Thus eluted RNA and DNA are highly concentrated and are ready to use for common downstream applications e g RT PCR PCR RNA size distribution RNA isolated from formalin fixed paraffin embedded tissue shows size distribution from 50 to 5 000 bases Often short sized RNA from ca 100 300 bases predominate especially when sample material is aged However samples which were subjected to good tissue fixation embedding and storage conditions can yield RNA even larger than 5 000 bases RNA integrity RNA Integrity Numbers RIN according to Agilent 2100 Bioanalyzer assays depend on sample type and quality In general RNA isolated from FFPE samples is poor in quality Typical RIN of RNA isolated with NucleoSpin FFPE kits are in the range of 2 6 rDNase is supplied with the kit DNA contaminations are removed by on column digestion with rDNase For most demanding downstream applications a subsequent digestion with rDNase in the eluate is possible see section 5 5 MACHEREY NAGEL 07 2010 Rev 02 7 Nucleic Acid Isolation from FFPE Samples Table 1 Ki
43. rade contaminating DNA This requires stringent RNase control and subsequent repurification of the RNA in order to remove buffer salts DNase and digested DNA The high quality RNase free recombinant DNase rDNase in the NucleoSpin FFPE kits facilitates such a digestion in solution in order to remove even traces of contaminat ing DNA A Digest DNA Reaction setup Prepare enzyme buffer premix Add 1 pl rDNase to 10 pl Reaction Buffer for rDNase Add 1 10 volume of enzyme buffer premix to the eluted RNA e g to 10 pl RNA add 1 ul of the premix comprising buffer and enzyme B Incubate for 10 min at 37 C Cc Inactivate rDNase After rDNase digestion in the RNA eluate incubate the sample for 5 min at 75 C to inactivate the rDNase Subsequently keep the sample on ice In most cases a further purification in order to remove inactivated rDNase buffer and salts is not necessary because the rDNase works in a highly dilute buffer and is inactivated during heat incubation If nevertheless a repurification is required NucleoSpin RNA Clean up XS is recommended see ordering information 36 MACHEREY NAGEL 07 2010 Rev 02 Nucleic Acid Isolation from FFPE Samples 6 Appendix 6 1 Comments on RNA quality and quantity Due to tissue fixation nucleic acids in FFPE samples are commonly fragmented and chemically modified by formaldehyde These modifications cannot be detected by stan dard quality contro
44. room temperature for 15 min 15 min It is not necessary to use a new Collection Tube after the incubation step 7 Wash and dry silica membrane 1 wash 100 pl FW1 Add 100 pl Buffer FW1 to the NucleoSpin FFPE RT Column Incubate for 2 min at RT 2 min Centrifuge for 30 s at 11 000 x g 2 11 000 x g Discard Collection Tube with flow through and place the 30s column into a new Collection Tube 2 ml Buffer FW1 will inactivate the rDNase 24 MACHEREY NAGEL 07 2010 Rev 02 RNA Isolation Paraffin Dissolver Add 400 jI Buffer FW2 to the NucleoSpin FFPE Column 400 pl FW2 Centrifuge for 30 s at 11 000 x g 11 000 x g Discard flow through and place the column back into the 30 s Collection Tube Add 200 jll Buffer FW2 to the NucleoSpin FFPE Column Centrifuge for 2 min at 11 000 x g to dry the membrane 200 pl FW2 Discard the Collection Tube with flow through and place e gt 11 000 x g the column into a nuclease free 1 5 ml Collection Tube 2 min provided If for any reason the liquid level in the Collection Tube reaches the NucleoSpin FFPE Column after centrifugation discard flow through and centrifuge again Elute highly pure RNA 10 pI H O Elute the RNA in 10 pl H O RNase free supplied and Aale centrifuge at 11 000 x g for 30 s If higher RNA concentration or higher elution volume is desired elution volume may be varied in the range of 11 000 x g 5 30 ul 30s MACHEREY NAG
45. sh and dry silica 4s 100 pl FW1 100 pl FW1 membrane RT 2 min RT 2 min O 11 000 x g 30 s 11 000 x g 30 s ou 400 pl FW2 400 pl FW2 cS 11 000 x g 30s 11 000 x g 30s gn 200 pl FW2 200 pl FW2 CD 11 000 x g 2 min 11 000 x g 2 min 8 Elute highly pure Qe 10 pl RNase free H O 10 ul RNase free H O RNA cS 11 000 x g 30s 11 000 x g 30s W MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 D 52355 D ren Germany Tel 49 0 24 21 969 270 www mn net com e mail tech bio mn net com RNA Isolation from FFPE Samples Protocol at a glance Rev 02 NucleoSpin FFPE RNA DNA Protocol 5 2 RNA isolation Protocol 5 4 RNA isolation with Paraffin Dissolver with xylene 1 Deparaffinize sample 400 ul Paraffin Dissolver 1 ml xylene 60 C 3 min RT 2 min Mix hot sample Mix Full speed 2 min Let sample cooldown Discard supernatant 1 ml 98 ethanol Mix Full speed 2 min Discard supernatant Dry at 60 C 3 10 min 2 Lyse sample 100 pl FL 100 pl FL eS 11 000 x g 1 min ji 10 pl Proteinase K 10 ul Proteinase K Mix Mix 60 C 15 min 3 hours 60 C 15 min 3 hours 3 Decrosslink 100 pl D Link 100 pl D Link Mix gently Mix gently 5 11 000 x g 30s _ 90 C 15 min 90 C 15 min 4 Adjust binding 200 pl 98 ethanol 200 pl 98 ethanol condition j Mix Mix eS 11 000 x g 30s 5 Bind RNA z Load aqueous lower phase Load lysate g J e5 2 000 x g 30 s 2 000 x g 3
46. t specifications at a glance Parameter NucleoSpin FFPE RNA DNA 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 Binding capacity 90 ug 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 usability of RNA and DNA obtained from FFPE samples The steps of tissue sampling post sampling delay before fixation fixation time embedding and storage conditions have high impact on RNA quality and yield For more details about these aspects see for example Chung JY et al 2008 van Maldegem F et al 2008 von Ahifen S et al 2007 Castiglione F et al 2007 Leyland Jones BR et al 2008 Starting from a paraffin embedded tissue block samples should be sectioned under RNase free conditions Paraffin sections may be stored at 4 C or lower for at least several weeks without observable effects on RNA yield or usability Long term storage of paraffin sections may have a negative effect on the RNA due to air oxidation Wear gloves at all times during the preparation Change gloves frequently When using the standard procedure with Paraffin Dissolver Processing larger quantities is possible with protocol modifications see section 2 4 8 MACHEREY NAGEL 07 2010 Rev 02 Nucleic Acid Isolation from FFPE Samples
47. ther information see Material Safety Data Sheet Disposal considerations for Paraffin Dissolver Please observe local regulations for collection and disposal of waste and contact waste disposal company where you will obtain information on disposal waste code number 16 05 06 MACHEREY NAGEL 07 2010 Rev 02 13 Nucleic Acid Isolation from FFPE Samples Risk phrases R10 R 20 21 22 R 22 R 36 38 R 36 37 38 R 42 R 42 43 R65 R 66 Flammable Harmful by inhalation in contact with the skin and if swallowed Harmful if swallowed Irritating to eyes and skin Irritating to eyes respiratory system and skin May cause sensitization by inhalation May cause sensitization by inhalation and skin contact Harmful May cause lung damage if swallowed Repeated exposure may cause skin dryness or cracking Safety phrases S16 S 22 S 24 S 26 S 36 37 S 62 Keep away from sources of ignition No smoking Do not breathe dust Avoid contact with the skin In case of contact with eyes rinse immediately with plenty of water and seek medical advice Wear suitable protective clothing and gloves If swallowed do not induce vomitting seek medical advice immediately and show this container or label 14 MACHEREY NAGEL 07 2010 Rev 02 RNA and DNA Isolation Paraffin Dissolver 5 Protocols NucleoSpin FFPE kits offer two different methods for sample deparaffinization One utilizes the Paraffin Dissolv
48. tions risk and safety phrases The following components of the NucleoSpin FFPE RNA DNA kits contain hazard ous contents Wear gloves and goggles and follow the safety instructions given in this section Component Hazard Hazard Risk Safety contents symbol phrases phrases rDNase rDNase x Xi May cause sensitiz R 42 43 S 22 24 RNase free lyophilized tion by inhalation and skin contact Proteinase K Proteinase K x Xn Irritating to eyes R 36 37 38 S 22 24 lyophilized Xi respiratory system 42 26 36 37 and skin May cause sensitization by inhalation FW1 Guanidinium x Xn Flammable R 10 22 S 7 16 hydrochloride Harmful if swallowed 36 38 ethanol Irritating to eyes lt 25 and skin MDB Guanidinium Flammable R10 S 7 16 thiocyanate lt 10 ethanol lt 10 Paraffin Mineral oil x Xn Harmful May cause R 65 66 S 62 Dissolver distillate lung damage if swal lowed Repeated exposure may cause skin dryness or cracking Hazard labeling not necessary if quantity per bottle below 25g or ml certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet Hazard labeling not necessary if quantity per bottle below 125g or ml certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 20 3 and TRGS 200 7 1 For fur
49. tissue pellets thus saving time NucleoSpin FFPE kits employ the odorless Paraffin Dissolver patent pending and allow 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 RNA and DNA into solution Subsequently heat incubation effectively eliminates crosslinking of DNA and RNA maintaining their integrity After addition of ethanol the lysate is applied to the NucleoSpin FFPE Column RNA and DNA are bound to the silica membrane After two washing steps DNA can be eluted in a low salt buffer Residual DNA remaining on the column is removed by convenient on column rDNase digestion RNase free rDNase is supplied with the kit Washing steps with two different buffers remove salts metabo lites and macromolecular cellular components Pure RNA is finally eluted under low ionic strength conditions in a small volume 10 ul of RNase free water yielding highly concentrated RNA Nucleic acid preparation using NucleoSpin FFPE kits can be performed at room tem perature The eluate however should be treated with care RNA is very sensitive to trace contaminations of RNases often found on general lab ware fingerprints and dust To ensure RNA stability keep RNA frozen at 20 C for short term or 70 C for long term storage DNA can be stored at 0 4 C for short term For long term storage
50. ven in section 3 rDNase solution not properly applied e Pipette rDNase solution directly onto the center of the silica membrane and close the lid in order to press the solution into the membrane Too much cell material used e Reduce quantity of cells or tissue used Contami nation of 7 RNA with DNA detection system too sensitive genomic e The amount of DNA contamination is significantly reduced dur DNA ing the on column digestion with rDNase Anyhow it can not be guaranteed that the purified RNA is 100 free of DNA therefore in very sensitive applications it might be possible to detect DNA The eventuality of DNA detection with PCR increases with the number of DNA copies per preparation single copy target lt plastidial mitochondrial target lt plasmid transfected into cells decrease in PCR amplicon size e Use larger PCR targets e g gt 500 bp or intron spanning prim ers if possible Use support protocol for subsequent rDNase digestion in the eluate section 5 5 Carry over of ethanol or salt e Do not let the column flow through touch the column outlet after the second Buffer FW2 wash Be sure to centrifuge at the rec Suboptimal ommended speed and time in order to remove ethanolic Buffer performance FW2 completely of RNA in e Check if Buffer FW2 has been equilibrated to room temperature downstream before use Washing at lower temperatures lowers efficiency of experiments salt removal by Buffer FW2 e D

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NucleoSpin® FFPE RNA/DNA