NucleoSpin® totalRNA FFPE XS - MACHEREY
Contents
1. Total RNA isolation from FFPE Samples User manual NucleoSpin totalRNA FFPE NucleoSpin totalRNA FFPE XS July 2015 Rev 04 MACHEREY NAGEL MN www mn net com Total RNA from FFPE samples Protocol at a glance Rev 04 page 1 1 Sample preparation NucleoSpin totalRNA FFPE Insert FFPE section s in a microcentrifuge tube NucleoSpin totalRNA FFPE XS Insert FFPE section s in a microcentrifuge tube 2 Deparaffinize sample 1 mL Paraffin Dissolver 56 C 5 min Vortex hot sample 16 000 x g 2 min 1 mL Paraffin Dissolver 56 C 5 min Vortex hot sample 16 000 x g 2 min 1 170 uLMLF 16 000 x g 2 min J 140 uL MLF 16 000 x g 2 min Remove Paraffin Dissolver Remove Paraffin Dissolver 3 Lyse sample A Quick protocol perform method 3A or 3B 15 uL Proteinase K 12 uL Proteinase K Mix gently Mix gently 56 C 15 min 56 C 15 min 15 uL MKA 12 uL MKA Vortex Vortex 0 C 5 min 0 C 5 min 16 000 x g 5 min 16 000 x g 5 min Transfer sample 80 C 15 min Transfer sample 80 C 15 min Lyse sample B Protocol for difficult to Iyse cells perform method 3A or 3B 15 uL Proteinase K 12 uL Proteinase K Mix gently Mix gently 56 C 90 min 56 C 90 min 15 uL MKA 12 uL MKA Vortex Vortex 0 C 5 min 0 C 5 min 16 000 x g 5 min 16 000 x g 5 min Transfer sample Transfer sample 4 Adjust binding condit2. 5 2 NucleoSpin totalRNA FFPE XS Before starting the preparation Check that rDNase and Buffer MW2 were prepared according to section 3 Set incubator s to 56 C for paraffin melting and lysis step and 80 C for decrosslink step Please note that lysis step 3A is the standard method for most common sample materials while lysis step 3B is utilized for difficult to lyse cells 1 Sample preparation Insert FFPE section s in a microcentrifuge tube not provided with the kit 2 Deparaffinize sample Add 1 mL Paraffin Dissolver to the sample Incubate 5 min at 56 C to melt the paraffin Vortex the hot sample Make sure that paraffin completely melts during the heat incubation step and mix well after melting to completely dissolve the paraffin Centrifuge sample for 2 min at 16 000 x g Attention Check Buffer MLF prior to use If a white precipitate is visible heat the buffer for several minutes at 30 40 C until the precipitate is completely dissolved and mix thoroughly Add 140 uL Buffer MLF Do not mix 6 n Centrifuge sample for 2 min at 16 000 x g Remove and discard Paraffin Dissolver by pipetting it off Note Slight residues of Paraffin Dissolver do not affect the following steps 3 A Lyse sample method A perform method 3A or 3 B Quick protocol Add 12 pL Proteinase K Mix by gently shaking or pipetting up and down Do not vortex 1 mL Paraffin Dissolver
3. C before use Poor sample quality Sample quality very much influences the obtainable RNA amount and quality For aspects concering sample harvest fixation embedding and storage refer to Castiglione F et al 2007 Chung J Y et al 2008 Leyland Jones B R et al 2008 von Ahlfsen S et al 2007 von Maldegem F et al 2008 Reagents not applied or restored properly Always dispense exactly the buffer volumes given in the protocols Always follow the given instructions closely with specific attention paid to order and mode of mixing shaking vortexing etc Add the indicated volume of 96 100 96 ethanol to Buffer MW2 Concentrate and mix thoroughly Store kit components at room temperature 18 25 C Storage at lower temperatures may cause salt precipitation If precipitation occurs incubate the bottle for several minutes at about 30 40 C and mix well until the precipitate is redissolved Keep bottles tightly closed in order to prevent evaporation or contamination 24 MACHEREY NAGEL 07 2015 Rev 04 Total RNA isolation from FFPE samples Poor RNA quality or yield continued lonic strength and pH influence Azg absorption as well as ratio A260 A280 For absorption measurement use 5 mM Tris pH 8 5 as diluent Please also see Manchester K L 1995 and Wilfinger W W et al 1997 Proteinase digestion time Depending of the nature of the sample an optimal digestion time
4. Get medical advice attention BEI Exposition oder falls betroffen Arztlichen Rat einholen rztliche Hilfe hinzuziehen Call a POISON CENTER doctor if you feel unwell Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen If skin irritation or rash occurs Get medical advice attention Bei Hautreizung oder ausschlag Arztlichen Rat einholen rztliche Hilfe hinzuziehen If eye irritation persists Get medical advice attention Bei anhaltender Augenreizung rztlichen Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTER doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM ArzV anrufen MACHEREY NAGEL 07 2015 Rev 04 13 Total RNA isolation from FFPE samples P363 P370 378 P403 233 P403 235 P405 Wash contaminated clothing before reuse Kontaminierte Kleidung vor erneutem Tragen waschen In case of fire Use to extinguish Bei Brand zum L schen verwenden Store in a well ventilated place Keep container tightly closed An einem gut bel fteten Ort aufbewahren Beh lter dicht verschlossen halten Store in a well ventilated place Keep cool An einem gut bel fteten Ort aufbewahren K hl halten Store locked up Unter Verschluss 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 A The symbol s
5. not provided sample 4 Adjust binding conditions 400 uL MX Add 400 uL Buffer MX and mix by vortexing 2 x 5 s Vortex Incubate for 1 min at room temperature 18 25 C RT 1 min 20 MACHEREY NAGEL 07 2015 Rev 04 NucleoSpin totalRNA FFPE XS 5 Bind RNA Place a NucleoSpin RNA FFPE XS Column in a new Load Collection Tube 2 mL sample Load sample onto the column and centrifuge for 15 s at 16 000 x g 9 ES 16 000 x g Discard flow through and place the column back into the 15s collection tube 6 Wash and dry silica membrane MW2 Add 400 pL Buffer MW2 to the NucleoSpin RNA FFPE XS Column Centrifuge for 15 s at 16 000 x g eS 16 000 x g Discard flow through and place the column back into the 155 collection tube Add 200 uL Buffer MW2 to the NucleoSpin RNA FFPE 200 uL XS Column MW2 Centrifuge for 1 min at 16 000 x g to dry the membrane letely Can 16 000 x g If the flow through in the collection tube has touched 1 min the NucleoSpin RNA FFPE XS Column after 2 wash discard flow through and centrifuge again 7 Optional Digest DNA Add 25 uL rDNase directly onto the silica membrane of 25 pL the NucleoSpin RNA FFPE XS Column rDNase Incubate at room temperature 18 25 C for 15 min RT 15 min Add 50 uL Buffer MX Incubate for 1 min at room temperature 18 25 C 50 uL MX Centrifuge for 15 s at 16 000 x g RT 1 min Discard flow through and place the column back into
6. 15 uL Buffer MKA and vortex briefly 15 uL MKA incubate ors mi Vortex ncubate for 5 min on ice 0 C 5 min Centrifuge for 5 min at 16 000 x g e 16 000 x g 5 min Transfer clear supernatant to a new 1 5mL Transfer microcentrifuge tube not provided sample 16 MACHEREY NAGEL 07 2015 Rev 04 NucleoSpin totalRNA FFPE 4 Adjust binding conditions Add 500 uL Buffer MX and mix by vortexing 2 x 5 s Incubate for 1 min at room temperature 18 25 C 5 Bind RNA Place a NucleoSpin RNA Column in a new Collection Tube 2 mL Load sample onto the column and centrifuge for 15 s at 16 000 x g Discard flow through and place the column back into the collection tube 6 Wash and dry silica membrane Add 700 uL Buffer MW2 to the NucleoSpin RNA Column Centrifuge for 15 s at 16 000 x g Discard flow through and place the column back into the collection tube Add 250uL Buffer MW2 to the NucleoSpin RNA Column Centrifuge for 1 min at 16 000 x g to dry the membrane completely If the flow through in the collection tube has touched the NucleoSpin RNA Column after 2 wash discard flow through and centrifuge again 7 Optional Digest DNA Add 50 pL rDNase directly onto the silica membrane of the NucleoSpin RNA Column Incubate at room temperature 18 25 C for 15 min 500 pL MX Vortex RT 1 min Load sample c5 16 000 x g 15s 700 uL MW2 gt 16
7. FFPE tissue Odorless and non toxic Paraffin Dissolver patent pending replaces the flammable and odorous xylene or d limonene commonly used for deparaffinization The tissue sample is then heat incubated with Proteinase K to digest the fixed tissue release nucleic acids and gently remove crosslinks Optimal binding conditions for even small RNA e g miRNA are adjusted and the lysate is applied to the NucleoSpin RNA Column NucleoSpin RNA FFPE XS Column RNA is bound to the silica membrane Residual DNA remaining on the membrane is removed by convenient on column rDNase digestion Washing steps remove salts metabolites and macromolecular cellular components Pure RNA is finally eluted in a small volume of RNase free water yielding highly concentrated RNA Nucleic acid preparation using NucleoSpin totalRNA FFPE XS can be performed at room temperature 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 MACHEREY NAGEL 07 2015 Rev 04 7 Total RNA isolation from FFPE samples 2 2 Kit specifications NucleoSpin totalRNA FFPE XS is recommended for the isolation of total RNA from formalin fixed paraffin embedded FFPE tissue samples typically as thin sections approx 3 20 um thickness Formalin fixed samples which
8. are not embedded in paraffin can also be used as sample material by omitting the deparaffinization steps The sample size can be up to 10 sections 1 20 uim of FFPE The amount of embedded tissue can be up to 50 mg for NucleoSpin totalRNA FFPE or up to 5 mg for NucleoSpin totalRNA FFPE XS 1x 10 um section with 1 cm tissue is approximately 1 mg RNA yield strongly depends on sample type quality and amount Furthermore the procedures of tissue sampling post sampling delay before fixation fixation time embedding and storage conditions have a high impact on RNA quality and yield For more details see for example Chung J Y et al 2008 van Maldegem F et al 2008 von Ahlfen S et al 2007 Castiglione F et al 2007 Leyland Jones B R et al 2008 RNA concentration RNA can be eluted highly concentrated and ready to use in a small volume of 30 50 uL NucleoSpin totalRNA FFPE or even 5 30 uL NucleoSpin totalRNA FFPE XS RNA size distribution RNA isolated from formalin fixed paraffin embedded tissue shows size distribution from 15 to 5 000 bases Often short sized RNA 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 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 gener
9. at lower temperatures may cause precipitation of salts If precipitation occurs incubate the bottle for several minutes at about 30 40 C and mix well until the precipitate is redissolved Before starting any NucleoSpin totalRNA FFPE XS protocol prepare the following RNase free rDNase Add the indicated volume of Reaction Buffer for rDNase 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 the enzyme too vigorously as rDNase is sensitive to mechanical agitation Dispense into aliquots and store at 20 C The frozen working solution is stable for at least 6 months Do not freeze thaw the aliquots more than three times Wash Buffer MW2 Add the indicated volumes of 96 100 ethanol to the MW2 concentrate Stored at room temperature 18 25 C the buffer is stable for at least one year NucleoSpin totalRNA FFPE 10 preps 50 preps 250 preps REF 740982 10 740982 50 740982 250 Wash Buffer MW2 6mL 25 mL 100 mL Concentrate Add 24 mL Add 100 mL Add 400 mL 96 100 96 ethanol 96 100 ethanol 96 100 ethanol RNase free 1 vial size A 1 vial size C 5 vials size C rDNase Add 0 75 mL Add 3 mL Add 3 mL lyophilized Reaction Buffer Reaction Buffer Reaction Buffer for rDNase for rDNase for rDNase to each vial 10 MACHEREY NAGEL 07 2015 Rev 04 Total RNA isolation from FFPE samples NucleoSpin totalRNA FFPE XS 10 preps 50 pr
10. 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 Last updated 07 2010 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 24 21 969 270 tech bio 2 mn net com 30 MACHEREY NAGEL 07 2015 Rev 04 MACHEREY NAGEL Germany and international Tel 49 24 21 969 0 E mail info mn net com MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 52355 D ren Germany Switzerland France USA MACHEREY NAGEL AG MACHEREY NAGEL EURL MACHEREY NAGEL Inc Tel 41 62 388 5500 Tel 33 388 68 22 68 Tel 1 484 821 0984 E mail sales ch mn net com E mail sales fr mn net com E mail sales us mn net com A045171 0750 5
11. the llection tube collection tube es 16 000 x g 15s MACHEREY NAGEL 07 2015 Rev 04 21 NucleoSpin totalRNA FFPE XS Add 400 uL Buffer MW to the NucleoSpin RNA FFPE 400 uL XS Column MW2 Centrifuge for 15 s at 16 000 x g Discard flow through and place the column back into the 16 000 x g collection tube 15s Add 200 uL Buffer MW to the NucleoSpin RNA FFPE 200 uL XS Column MW2 Centrifuge for 1 min at 16 000 x g Discard flow through and place the column back into the 16 000 x g collection tube 1 min Centrifuge for 5 min at 16 000 x gto dry the membrane C 16 000 x g 5 min 8 Elute highly pure RNA Place the NucleoSpin RNA FFPE XS Column in a new 5 30 uL Collection Tube 1 5 mL RNase free H Add 5 pL for high concentration to 30 pL for high 0 yield RNase free H O to the column RT 1 min Incubate for 1 min at room temperature 18 25 C es 16 000 x g Centrifuge for 1 min at 16 000 x g Y min Keep the eluted RNA on ice or freeze at 20 C short term storage or 70 C long term storage 22 MACHEREY NAGEL 07 2015 Rev 04 DNA digestion in the RNA eluates 5 3 DNA digestion in the RNA eluates Comments on DNA removal Although the on column rDNase digest in the standard protocol is very efficient there are still certain applications which require even lower quantities of residual DNA For example RT PCR reactions with primers that do not differenti
12. 000 x g 15s 250 uL MW2 C 16 000 x g 1 min 50 uL rDNase RT 15 min MACHEREY NAGEL 07 2015 Rev 04 17 NucleoSpin totalRNA FFPE Add 100 uL Buffer MX Incubate for 1 min at room temperature 18 25 C Centrifuge for 15 s at 16 000 x g Discard flow through and place the column back into the collection tube Add 700 uL Buffer MW2 to the NucleoSpin RNA Column Centrifuge for 15 s at 16 000 x g Discard flow through and place the column back into the collection tube Add 250 uL Buffer MW2 to the NucleoSpin RNA Column Centrifuge for 1 min at 16 000 x g to dry the membrane completely If the flow through in the collection tube has touched the NucleoSpin RNA Column after 2 wash discard flow through and centrifuge again Elute highly pure RNA Place the NucleoSpin RNA Column in a new Collection Tube 1 5 mL Add 30 uL for high concentration or 50 uL for high yield RNase free H O to the column Incubate for 1 min at room temperature 18 25 C Centrifuge for 1 min at 16 000 x g Keep the eluted RNA on ice or freeze at 20 C short term storage or 70 C long term storage 16 000 x g 15s 100 uL MX RT 1 min c5 700 uL MW2 e 16 000 x g 15s 250 uL MW2 eS 16 000 x g 1 min 30 50 uL RNase free H O RT 1 min cS 16 000 x g 1 min MACHEREY NAGEL 07 2015 Rev 04 NucleoSpin totalRNA FFPE XS
13. 56 C 5 min Vortex hot sample e 16 000 x g 2 min 140 uL MLF CS 16 000 x g 2 min Remove Paraffin Dissolver 12 uL Proteinase K Mix gently MACHEREY NAGEL 07 2015 Rev 04 19 NucleoSpin totalRNA FFPE XS Incubate for 15 min at 56 C to lyse sample tissue If tissue is still visible continue incubation until sample 56 C 15 min is digested 12 uL MKA Add 12 pL Buffer MKA and vortex briefly Vortex Incubate for 5 min on ice 0 C 5 min Centrifuge for 5 min at 16 000 x g es 16 000 x g 5 min Transfer clear supernatant to a new 1 5mL microcentrifuge tube not provided Transfer Incubate at 80 C for exactly 15 min sample Shorter incubation or lower temperature leads to 80 C 15 min incomplete decrosslinking Longer incubation might lead to degradation of RNA 3 B Lyse sample method B perform method 3A or 3 B Protocol for diffcult to lyse 12 pL samples I Proteinase K Add 12 pL Proteinase K Mix gently Mix by gently shaking or pipetting up and down Do not 56 C 90 min vortex Incubate for 90 min at 56 C to lyse sample tissue If tissue is still visible continue increase incubation time up to overnight until sample is digested 12 uL MKA Add 12 pL Buffer MKA and vortex briefly Vortex Incubate for 5 min on ice 0 C 5 min Centrifuge for 5 min at 16 000 x g e 16 000 x g 5 min Transfer clear supernatant to a new 1 5mL Transfer microcentrifuge tube
14. DANGER 337 313 GEFAHR 370 378 403 233 403 235 405 Hazard phrases H225 Highly flammable liquid and vapour Fl ssigkeit und Dampf leicht entz ndbar H317 May cause an allergic skin reaction Kann allergische Hautreaktionen verursachen H319 Causes serious eye irritation Verursacht schwere Augenreizung H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursachen H335 May cause respiratory irritation Kann die Atemwege reizen 12 MACHEREY NAGEL 07 2015 Rev 04 Total RNA isolation from FFPE samples H351 EUH019 EUHO66 Suspected of causing cancer state route of exposure if it is conclusively proven that no other routs of exposure cause the hazard Kann vermutlich Krebs erzeugen Expositionsweg angeben sofern schl ssig belegt ist dass diese Gefahr bei keinem anderen Expositionsweg besteht May form explosive peroxides Kann explosionsf hige Peroxide bilden Repeated exposure may cause skin dryness or cracking Wiederholter Kontakt kann zu spr der oder rissiger Haut f hren Precaution phrases P201 P202 P210 P261 P264 P271 P272 P280 P302 352 P304 340 P305 351 338 P308 313 P312 P333 313 P337 313 P342 311 Obtain special instructions before use Vor Gebrauch besondere Anweisungen einholen Do not handle until all safety precautions have
15. Histochemistry amp Cytochemistry 56 11 1033 1042 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 Manchester K L 1995 Value of Azgo Azgp ratios for measurement of purity of nucleic acids Biotechniques 19 208 209 von Ahlfen S et al 2007 Determinants for RNA quality from FFPE samples PLoS ONE Issue 12 e1261 von Maldegem F et al 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 Wilfinger W W et al 1997 Effect of pH and ionic strength on the spectorophotometric assessment of nucleic acid purity Biotechniques 22 474 481 28 MACHEREY NAGEL 07 2015 Rev 04 Total RNA isolation from FFPE samples 6 4 Product use restriction warranty NucleoSpin totalRNA FFPE NucleoSpin totalRNA 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 refe
16. SS 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 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 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
17. al the quality of RNA extracted from FFPE samples is poor Typical RIN of RNA isolated with NucleoSpin totalRNA FFPE XS kits are in range of 2 6 rDNase is supplied with the kit for a convenient removal of DNA by on column digestion For more demanding downstream applications DNA can also be digested in solution as described in section 5 2 8 MACHEREY NAGEL 07 2015 Rev 04 Total RNA isolation from FFPE samples Table 1 Kit specifications at a glance NucleoSpin NucleoSpin Parameter totalRNA FFPE totalRNA FFPE XS Technology Silica membrane technology Silica membrane technology Format Mini spin columns Mini spin columns Sample material Typical yield Elution volume Preparation time Up to 10 sections with up to 50 mg of tissue Strongly depends on sample quality and amount 30 50 uL 70 min 6 preps 90 min including optional rDNase digest XS design Up to 10 sections with up to 5 mg of tissue Strongly depends on sample quality and amount 5 30 uL 70 min 6 preps 90 min including optional rDNase digest MACHEREY NAGEL 07 2015 Rev 04 Total RNA isolation from FFPE samples 3 Storage conditions and preparation of working solutions Storage conditions Store lyophilized rDNase and Proteinase K at 4 C upon arrival stable for at least 1 year All other kit components should be stored at room temperature 18 25 C and are stable for at least one year Storage
18. ate between cDNA derived from RNA and contaminating genomic DNA DNA digestion in solution can efficiently degrade contaminating DNA This requires stringent RNase control and optionally repurification of the RNA in order to remove buffer salts DNase and digested DNA The high quality RNase free recombinant DNase rDNase provided with the kit facilitates such a digestion in solution A Digest DNA Reaction setup Add 1 10 volume of rDNase dissolved in Reaction Buffer for rDNase to the eluted RNA e g add 3 uL enzyme to 30 uL RNA eluate B Incubate for 10 min at 37 C C Inactivate rDNase Incubate the sample for 5 min at 75 C to inactivate the rDNase Put the sample on ice In most cases a further purification in order to remove inactivated rDNase buffer and salts is not necessary If nevertheless a repurification is required NucleoSpin RNA Clean up XS is recommended see ordering information MACHEREY NAGEL 07 2015 Rev 04 23 Total RNA isolation from FFPE samples 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Poor RNA quality or yield RNase contamination Create an RNase free working environment Wear gloves during all steps of the procedure Change gloves frequently Use sterile disposable polypropylene tubes and filter strips Keep tubes closed whenever possible during the preparation Glassware should be oven baked for at least 2 hours at 250
19. been read and understood Vor Gebrauch alle Sicherheitsratschl ge lesen und verstehen Keep away from heat hot surfaces sparks open flames and other ignition sources No smoking Von Hitze heissen Oberfl chen Funken offenen Flammen sowie anderen Z ndquellenarten fernhalten Nicht rauchen Avoid breathing dust fume gas mist vapours spray Einatmen von Staub Rauch Gas Nebel Dampf Aerosol vermeiden Wash thoroughly after handling Nach Handhabung gr ndlich waschen Use only outdoors or in a well ventilated area Nur im Freien oder in gut bel fteten R umen verwenden Contaminated work clothing should not be allowed out of the workplace Kontaminierte Arbeitskleidung nicht auBerhalb des Arbeitsplatzes tragen Wear protective gloves protective clothing eye protection face protection Schutzhandschuhe Schutzkleidung Augenschutz Gesichtsschutz tragen IF ON SKIN Wash with plenty of water BEI BER HRUNG MIT DER HAUT Mit viel Wasser waschen IF INHALED Remove person to fresh air and keep comfortable for breathing BEI EINATMEN Die Person an die frische Luft bringen und f r ungehinderte Atmung sorgen IF IN EYES Rinse cautiously with water for several minuts Remove contact lenses if present and easy to do Continue rinsing BEI KONTAKT MIT DEN AUGEN Einige Minuten lang behutsam mit Wasser aussp len Eventuell vorhandene Kontaktlinsen nach M glichkeit entfernen Weiter aussp len IF exposed or concerned
20. e robustness of the RT PCR system used RT PCR might be inhibited if complete eluates are used as template for RT PCR Use less eluate as template 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 Ago quantification values and PCR quantification 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 quantification via Ag 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 0 Quantification of small RNA amounts centrifuge the eluate for 30 s at 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 RiboGreen fluorescent dye Unexpected Aaeo Aago ratio Measurement not in the range of photometer detection limit In order to obtain a reliable A go Aag ratio it is necessary that the initially measured Ago and Ago values a
21. eps 250 preps REF 740982 10 740982 50 740982 250 Wash Buffer MW2 6mL 25 mL 100 mL Concentrate Add 24 mL Add 100 mL Add 400 mL 96 100 ethanol 96 100 ethanol 96 100 ethanol RNase free 1 vial size A 1 vial size C 3 vials size C rDNase Add 0 75 mL Add 3 mL Add 3 mL lyophilized Reaction Buffer Reaction Buffer Reaction Buffer for rDNase for rDNase for rDNase to each vial MACHEREY NAGEL 07 2015 Rev 04 11 Total RNA isolation from FFPE samples 4 Safety instructions The following components of the NucleoSpin totalRNA 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 or 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden GHS Hazard Precaution Component Hazard contents symbol phrases phrases Inhalt Gefahrstof GHS Symbol H S tze P S tze Liquid Proteinase K 1 3 317 261 272 280 Proteinase K Proteinase K 1 3 302 352 333 313 363 CAS 39450 0 1 61 WARNING ACHTUNG rDNase rDNase 90 100 317 334 261 272 280 rDNase 90 100 lt gt 302 352 304 340 CAS 9003 98 9 DANGER 333 313 GEFAHR 342 311 363 MX Dioxane 40 90 225 319 201 202 210 Dioxan 40 90 335 351 261 264 271 EUHO019 280 304 340 zz EUHO66 305 351 338 308 313 312
22. 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 6 MACHEREY NAGEL 07 2015 Rev 04 Total RNA isolation from FFPE samples 2 Product description Formalin fxed and paraffin embedded FFPE tissue is commonly used in histopathological analysis Recently there is more and more interest in also investigating DNA modifications RNA expression or miRNA profiles of old archived FFPE samples However fixation embedding and storage lead to crosslinking and fragmentation of RNA Especially crosslinks cannot be detected by standard quality control assays such as gel electrophoresis spectrophotometry or microfluidic analysis but the efficiency of enzymatic reactions is significantly reduced for example in RT PCR Standard RNA purification procedures do not remove these chemical modifications and therefore result in low RNA yield or poor downstream application performance The NucleoSpin totalRNA FFPE XS procedure implements buffers and procedural steps to efficiently decrosslink nucleic acids and yield high quality RNA for the most demanding applications 2 1 The basic principle The NucleoSpin totalRNA FFPE XS kits provide a convenient reliable and fast method to isolate RNA from formalin fixed paraffin embedded
23. from 15 min to 3 hours has to be determined empirically If tissue residues are still visible after 15 min continue the incubation for up to 3 hours If a large portion of the sample still remains undigested continue digestion overnight An overnight incubation is not recommended if the tissue digested well within 3 hours Contami nation of RNA with genomic DNA rDNase not active e Reconstitute and store lyophilized rDNase according to instructions given in section 3 rDNase solution not properly applied 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 Reduce quantity of cells or tissue used Use larger PCR targets e g gt 500 bp or intron spanning primers for RNA analysis Use support protocol for subsequent rDNase digestion in the eluate section 5 2 MACHEREY NAGEL 07 2015 Rev 04 25 Total RNA isolation from FFPE samples Suboptimal performance of RNA in downstream experiments Carry over of ethanol or salt Do not let the column flow through touch the column outlet after the second Buffer MW2 wash Be sure to centrifuge at the recommended speed and time in order to remove ethanolic Buffer MW2 completely Check that Buffer MW2 has been equilibrated to room temperature before use Washing at lower temperatures lowers efficiency of salt removal Depending on th
24. g Be 9 Remove and discard Paraffin Dissolver by pipetting it Remove off Paraffin Note Slight residues of Paraffin Dissolver do not affect Dissolver the following steps MACHEREY NAGEL 07 2015 Rev 04 15 NucleoSpin totalRNA FFPE 3 A Lyse sample method A perform method 3A or 3B Quick protocol 15 pL Add 15 pL Proteinase K Proteinase K Mix by gently shaking or pipetting up and down Do not Mix gently vorax 56 C 15 min Incubate for 15 min at 56 C to lyse sample tissue If tissue is still visible continue incubation until sample is digested Add 15 uL Buffer MKA and vortex briefly 15 uL MKA i c bate formi Vortex ncubate for 5 min on ice 0 C 5 min Centrifuge for 5 min at 16 000 x g e 16 000 x g 5 min Transfer clear supernatant to a new 1 5mL microcentrifuge tube not provided Transfer Incubate at 80 C for exactly 15 min sample Shorter incubation or lower temperature leads to 80 C 15 min incomplete decrosslinking Longer incubation might lead to degradation of RNA 3 B Lyse sample method B perform method 3A or 3 B Protocol for diffcult to lyse 15 pL samples P Proteinase K Add 15 pL Proteinase K eu 1 Mix gently Mix by gently shaking or pipetting up and down Do not 56 C 90 min vortex Incubate for 90 min at 56 C to lyse sample tissue If tissue is still visible continue increase incubation time up to overnight until sample is digested Add
25. hown on labels refers to the precaution phrases of this section Das auf Etiketten dargestellte Symbol weist auf die P S tzen dieses Kapitels hin 14 MACHEREY NAGEL 07 2015 Rev 04 NucleoSpin totalRNA FFPE 5 Protocols 5 1 NucleoSpin totalRNA FFPE Before starting the preparation Check that rDNase and Buffer MW2 were prepared according to section 3 Set incubator s to 56 C for paraffin melting and lysis step and 80 C for decrosslink step Please note that lysis step 3A is the standard method for most common sample materials while lysis step 3B is utilized for difficult to lyse cells 1 Sample preparation Insert FFPE section s in a microcentrifuge tube not provided with the kit 2 Deparaffinize sample Add 1 mL Paraffin Dissolver to the sample 1mL Incubate 5 min at 56 C to melt the paraffi Foren ncubate 5 min a to melt the paraffin Dissolvor Vortex the hot sample 56 C 5 min Make sure that paraffin completely melts during the heat V i ortex incubation step and mix well after melting to completely i 5 hot sample dissolve the paraffin Centrifuge sample for 2 min at 16 000 x g c U 9 Attention Check Buffer MLF prior to use If a white precipitate is visible heat the buffer for several minutes _ 170 uL at 30 40 C until the precipitate is completely dissolved ML and mix thoroughly Add 170 uL Buffer MLF Do not mix Centrifuge sample for 2 min at 16 000 x
26. ions aja T T en 6 en 500 uL MX Vortex RT 1 min em j j ef 1 a4 Ca af 400 uL MX Vortex RT 1 min 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 Total RNA from FFPE samples Protocol at a glance Rev 04 page 2 NucleoSpin totalRNA FFPE NucleoSpin totalRNA FFPE XS 5 Bind RNA Load sample Load sample 16 000 x g 15s 16 000 x g 15s 6 Wash and dry silica 700 uL MW2 400 uL MW2 membrane 16 000 x g 15s 16 000 x g 15s 250 uL MW2 250 uL MW2 16 000 x g 1 min 16 000 x g 1 min 7 Optional Digest DNA 50 uL rDNase 25 uL rDNase RT 15 min RT 15 min 100 uL MX 50 uL MX RT 1 min RT 1 min 16 000 x g 15s 16 000 x g 15s 700 uL MW2 400 uL MW2 16 000 x g 15s 16 000 x g 15s 250 uL MW2 200 uL MW2 16 000 x g 1 min 16 000 x g 1 min 8 Elute highly 30 50 uL 30 50 uL pure RNA RNase free H O RNase free H O RT 1 min RT 1 min 16 000 x g 1 min 16 000 x g 1 min 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 Total RNA isolation 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 6 1 3 About this user manual 6 2 Produc
27. ll 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 specifications 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 MACHEREY NAGEL 07 2015 Rev 04 29 Total RNA isolation from FFPE samples 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 FITNE
28. r 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 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 expressly 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 sha
29. re significantly above the detection limit of the photometer used An Aggy value close to the background noise of the photometer will cause non reliable Aggo Aago ratios 26 MACHEREY NAGEL 07 2015 Rev 04 Total RNA isolation from FFPE samples 6 2 Ordering information Product REF NucleoSpin totalRNA FFPE 740982 10 50 250 NucleoSpin totalRNA FFPE XS 740969 10 50 250 NucleoSpin RNA XS 740902 10 50 250 NucleoSpin RNA Clean up XS 740908 10 50 250 NucleoSpin RNA 740955 10 50 250 NucleoSpin RNA Protein 740933 10 50 250 NucleoSpin TriPrep 740966 10 50 250 rDNase Set 740963 Paraffin Dissolver 740968 25 Collection Tubes 2 mL 740600 Visit www mn net com for more detailed product information Pack of 10 50 250 preps 10 50 250 preps 10 50 250 preps 10 50 250 preps 10 50 250 preps 10 50 250 preps 10 50 250 preps 25 mL 1000 DISTRIBUTION AND USE IN THE USA IS PROHIBITED FOR PATENT REASONS MACHEREY NAGEL 07 2015 Rev 04 27 Total RNA isolation from FFPE samples 6 3 References Castiglione F et al 2007 Real time PCR analysis of RNA extracted from formalin fixed and 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
30. solver 15 mL 60 mL 300 mL Lysis Buffer MLF 10 mL 10 mL 50 mL Precipitation Buffer MKA 1mL 1mL 10 mL Binding Buffer MX 13 mL 60 mL 250 mL Reaction Buffer for rDNase 7 mL 7 mL 30 mL Wash Buffer MW2 6 mL 25 mL 100 mL Concentrate RNase free H O 13 mL 13 mL 13 mL rDNase RNase free 1 vial 1 vial 3 vials Iyophilized size A size C size C Liquid Proteinase K 0 2 mL 0 8 mL 3 x 1 25 mL NucleoSpin RNA FFPE XS 10 50 250 Columns light blue rings Collection Tubes 2 mL 10 50 250 Collection Tubes 1 5 mL 10 50 250 User manual 1 1 1 For preparation of working solutions and storage conditions see section 3 MACHEREY NAGEL 07 2015 Rev 04 5 Total RNA isolation from FFPE samples 1 2 Reagents consumables and equipment to be supplied by user Reagents 96 100 ethanol preferably undenaturated ethanol Consumables 1 5 mL microcentrifuge tubes for sample lysis Disposable pipette tips Equipment Manual pipettors Centrifuge for microcentrifuge tubes Vortex mixer Thermal heating block adjustable to 56 C and 80 C Personal protection equipment lab coat gloves goggles 1 3 About this user manual It is strongly recommended that first time users of NucleoSpin totalRNA 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 glance is designed to be used only as a supplemental tool
31. t description 7 2 1 The basic principle 7 2 2 Kit specifications 8 Storage conditions and preparation of working solutions 10 4 Safety instructions 12 5 Protocols 15 5 1 NucleoSpin totalRNA FFPE 15 5 2 NucleoSpin totalRNA FFPE XS 19 5 3 DNA digestion in the RNA eluates 238 6 Appendix 24 6 1 Troubleshooting 24 6 2 Ordering information 27 6 3 References 28 6 4 Product use restriction warranty 29 MACHEREY NAGEL 07 2015 Rev 04 3 Total RNA isolation from FFPE samples 1 Components 1 1 Kit contents NucleoSpin totalRNA FFPE 10 preps 50 preps 250 preps REF 740982 10 740982 50 740982 250 Paraffin Dissolver 15 mL 60 mL 300 mL Lysis Buffer MLF 10 mL 10 mL 50 mL Precipitation Buffer MKA 1mL 1mL 10 mL Binding Buffer MX 13mL 60 mL 250 mL Reaction Buffer for rDNase 7 mL 7 mL 30 mL Wash Buffer MW2 6 mL 25mL 100 mL Concentrate RNase free H O 13 mL 13 mL 13 mL rDNase RNase free 1 vial 1 vial 5 vials Iyophilized size A size C size C Liquid Proteinase K 0 6 mL 0 8 mL 5mL NucleoSpin RNA Columns 10 50 250 light blue rings Collection Tubes 2 mL 10 50 250 Collection Tubes 1 5 mL 10 50 250 User manual 1 1 1 For preparation of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 07 2015 Rev 04 Total RNA isolation from FFPE samples 1 1 Kit contents continued NucleoSpin totalRNA FFPE XS 10 preps 50 preps 250 preps REF 740969 10 740969 50 740969 250 Paraffin Dis
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