Small RNA and DNA from Plasma User manual
Contents
1. Small RNA and DNA from Plasma User manual NucleoSpin miRNA Plasma July 2014 Rev 03 MACHEREY NAGEL MN www mn net com Isolation of miRNA from plasma Protocol at a glance Rev 03 NucleoSpin miRNA Plasma 1 Prepare sample 300 uL plasma or serum 90 uL MLP Vortex 5 s RT 3 min 2 Precipitate protein 30 uL MPP E Vortex 5 s RT 1 min Y 11 000 x g 3 min gt 3 Transfer supernatant eB Transfer clear supernatant to Collection Tube 2 mL lid Yv 4 Adjust binding 400 uL isopropanol conditions k Vortex 5 s Y 5 Bind RNA and DNA S Load sample on NucleoSpin miRNA Column E eS RT 2 min 6 we 11 000 x g 30s 6 Optional Optional Digest DNA 2 700 pLMW2 11 000 x g 30 s E z0ou mw2 11 000x g 2 min 50 uL rDNase in Reaction Buffer for rDNase RT 15 min 7 Wash and dry 100 uLMW1 11 000 x g 30s 700 uL MW2 11 000 x g 30 s 250 uLMW2 11 000 x g 2 min 8 Elute RNA 30 uL RNase free H O RT 1 min 11 000 x g 1 min Larger sample volumes can be processed when buffer volumes of MLP MPP and isopropanol are increased proportionally Multiple loading is necessary in step 5 see section 2 3 for more information 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 Small RNA and DNA from plasma Table of contents 1 Co2. at lower temperatures may cause salt precipitation Heat buffer with precipitated salt to 30 C until salt is dissolved Let the buffer cool down to room temperature before use Keep bottles tightly closed in order to prevent evaporation or contamination 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 tips Keep tubes closed whenever possible during the preparation unless stated otherwise Glassware should be oven baked for at least 2 hours at 250 C before use Suboptimal performance of RNA in downstream experiments Inhibition by co purified RT PCR inhibitors Heme hemin and other degradation products of red blood cells strongly inhibit reverse transcription and PCR Too much plasma or bad plasma quality can result in contamination with these inhibitors Use less plasma dilute eluates perform the optional Proteinase K digest see section 2 4 or add BSA prior to RT or PCR reactions 16 MACHEREY NAGEL 07 2014 Rev 03 Small RNA and DNA from plasma Possible cause and suggestions Do not let the flow through touch the column outlet after the second MW2 wash Make sure to centrifuge at the corresponding speed for the respective time in order to remove ethanolic buffer Check if buffer MW2 has been equilibrated to room temperature 18 25 C before use Washing at
3. miRNA Column Centrifuge for 30 s at 11 000 x g Discard flow through and place the column back into the collection 11 000 x g tube 30 s Add 250 uL Buffer MW2 to the NucleoSpin miRNA 250 pL MW2 Column Centrifuge for 2 min at 11 000 x g It is not necessary to discard the flow through 11 000 x g 2 min Digest DNA i 50 pL Add 50 uL rDNase dissolved in Reaction Buffer for rDNase rDNase according to section 3 directly onto the silica membrane of the NucleoSpin miRNA Column RT Close the lid and incubate at room temperature 18 15 min 25 C for 15 min MACHEREY NAGEL 07 2014 Rev 03 13 NucleoSpin miRNA Plasma 7 Wash and dry silica membrane 100 pL MW1 Add 100 uL Buffer MW1 to the NucleoSpin miRNA l trifuge f t 11 Column Centrifuge for 30 s at 11 000 x g 11 000 x g Discard flow through and place the column back into the 30s collection tube Add 700 pL Buffer MW2 to the NucleoSpin miRNA 700 uL MWe Column Centrifuge for 30 s at 11 000 x g Discard flow through and place the column back into the 11 000 x g collection tube 30 s Add 250 uL Buffer MW2 to the NucleoSpin miRNA Column Centrifuge for 2 min at 11 000 x g to dry the membrane 250 pL MW2 completely If the liquid in the collection tube has touched the NucleoSpin miRNA Column after the 3 wash discard 11 000 xg flow through and centrifuge again 2 min Note The same collection tube is used throughout the entire
4. symptoms Call a POISON CENTER doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM Arzt anrufen Wash contaminated clothing before reuse Kontaminierte Kleidung vor erneutem Tragen waschen Store in a well ventilated place Keep cool An einem gut bel fteten Ort lagern K hl halten 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 MACHEREY NAGEL 07 2014 Rev 03 11 NucleoSpin miRNA Plasma 5 Protocol small RNA and DNA purification from plasma or serum Before starting with the preparation check that isopropanol is available that ethanol was added to Wash Buffer MW2 and that rDNase was reconstituted according to section 3 1 Prepare sample Note See section 2 4 for optional Proteinase K digest Add 90 uL Buffer MLP to 300 pL sample 90 pL MLP Vortex for 5 s Vortex 5s Incubate for 3 min at room temperature 18 25 C RT 3 min Note To process 600 uL or 900 uL sample material increase volumes for Buffer MLP MPP and isopropanol proportionally Multiple loading steps will be necessary in step 5 See section 2 3 for more information 2 Precipitate protein 30 pL MPP Add 30 uL Buffer MPP and vortex for 5 s 5 Vortex 5s Incubate for 1 min at room temperature 18 25 C RT 1 min Centrifuge for 3 min at 11 000 x g to pellet the protein es 11 000 x g 3 m
5. washing procedure to reduce plastic waste If new collection tubes are to be used for each step see ordering information 14 MACHEREY NAGEL 07 2014 Rev 03 NucleoSpin miRNA Plasma Elute RNA and DNA Place the NucleoSpin miRNA Column in a new Collection Tube 1 5 mL Note The elution buffer volume highly influences the final yield and concentration and furthermore influences elution efficiency of large oligonucleotides See section 2 6 for more information about elution in 20 30 or 50 uL Add 30 uL RNase free H O directly onto the silica membrane of the column Incubate for 1 min at room temperature 18 25 C Close the lid and centrifuge for 1 min at 11 000 x g 30 pL RNase free F H O RT 1 min 11 000 x g 1 min MACHEREY NAGEL 07 2014 Rev 03 15 Small RNA and DNA from plasma 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Poor or no RNA yield RNA is degraded Reagents not applied or restored properly Always dispense exactly the buffer volumes given in the protocols The correct proportions of buffers MLP MPP and isopropanol are essential for optimal yield and purity Always follow closely the given instructions with regard to order and mode of mixing shaking vortexing etc Add the indicated volume of 96 100 ethanol to Buffer MW2 Concentrate and mix thoroughly Store kit components at room temperature 18 25 C Storage
6. 02 352 P 304 340 P 330 P 333 313 P 342 311 P 363 P 403 235 Keep away from heat hot surfaces sparks open flames and other ignition sources No smoking Von Hitze hei en Oberfl chen Funken offenen Flammen sowie anderen Z ndquellenarten fernhalten Nicht rauchen Keep container tightly closed Beh lter dicht verschlossen halten Do not breathe dust fume gas mist vapours spray Staub Rauc Gas Nebel Dampf Aerosol nicht einatmen Avoid breathing dust Einatmen von Staub vermeiden Contaminated work clothing should not be allowed out of the workplace Kontaminierte Arbeitskleidung nicht au erhalb des Arbeitsplatzes tragen Avoid release to the environment Freisetzung in die Umwelt vermeiden Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen IF SWALLOWED Call a POISON CENTER doctor if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen IF ON SKIN Wash with plenty of water BEI KONTAKT MIT DER HAUT Mit viel Wasser waschen IF INHALED Remove victim to fresh air and keep at rest in a position com fortable for breathing BEI EINATMEN An die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert Rinse mouth Mund aussp len If skin irritation occurs Get medical advice attention Bei Hautreizung rztlichen Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory
7. AL LABORATORY USE ONLY MACHEREY NAGEL products are suited for QUALIFIED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet of the product MACHEREY NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products in other fields of application Application on the human body is STRICTLY FORBIDDEN The respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for IN VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for IN VITRO diagnostic use Please pay attention to the package of the product IN VITRO diagnostic products are expressiy marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR IN VITRO DIAGNOSTIC USE ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE No claim or representations is intended for its use to identify any specific organism or for clinical use included but not limited to diagnostic prognostic therapeutic or blood banking It is rather in the responsibility of the user or in any case of
8. FICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit 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 Prod
9. arlier signal in qRT PCR which is rather insignificant for detection sensitivity compared to the much larger differences that occur from sample to sample or between different miRNAs Furthermore if plasma quality is low co purified RT PCR inhibitors might require diluting the eluate and thereby counteract the increased yields Figure 1 shows qRT PCR results from eight different plasma samples analyzed for miR 16 very high expression and miR 1 very low expression They differ in average by about 14 cycles which is a difference in expression by 3 4 orders of magnitude 1 000 10 000 fold Doubling the amount of sample material would just shift the mean values from 32 6 to 31 6 miR 1 and from 18 3 to 17 3 miR 16 This is much less than the variation from sample to sample and can thus be neglected 35 33 31 29 27 25 qRT PCR Ct 23 miR 1 miR 16 Figure 1 qRT PCR quantification of miR 1 and miR 16 MIRNA from only 300 uL of eight different blood plasma samples were purified and eluted in 30 uL RNase free water 2 uL of each eluate were used for a 10 uL RT reaction Applied Biosystems TaqMan MicroRNA RT Kit with miRNA specific primers Applied Biosystems hsa miR 1 MicroRNA Assay and hsa miR 16 MicroRNA Assay The RT reaction mix was diluted 1 10 Only 4 uL of the resulting 100 uL were used for the PCR reaction Applied Biosystems TaqMan Universal PCR Master Mix in combination with the MicroRNA Assays for
10. e digest The remaining nucleic acids are washed and eluted with minimal amounts of RNase free water 2 2 Kit specifications The NucleoSpin miRNA Plasma kit is designed for the isolation of short RNA and DNA lt 1000 nt bp from cell free blood plasma or serum rDNase is provided for an optional on column digest to remove traces of genomic DNA The eluted RNA and DNA are ready to use for all standard downstream applications for example qPCR qRT PCR Northern Blot chip hybridization Table 1 Kit specifications at a glance Parameter NucleoSpin miRNA Plasma Sample size 300 uL blood plasma or serum Fragment size lt 1000 nt op Binding capacity 200 ug Elution volume 20 50 uL Preparation time 40 min 10 preps without rDNase digest 70 min 10 preps with rDNase digest 6 MACHEREY NAGEL 07 2014 Rev 03 Small RNA and DNA from plasma 2 3 Amount of starting material The standard procedure allows to process 300 uL of sample material with only one loading step onto the NucleoSpin miRNA Column This is usually enough to detect also low abundance miRNA in plasma or serum If larger sample volumes are to be used to increase the sensitivity even further the volumes of Buffer MLP and Buffer MPP as well as the isopropanol have to be increased proportionally Multiple loading steps per sample are necessary But consider that doubling or tripling the starting volume will result in an only 1 1 5 cycles e
11. e the following Wash Buffer MW2 Add the indicated volumes of 96 100 ethanol to the MW2 concentrate The buffer can be stored at room temperature 18 25 C for at least one year 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 rDNase vigorously as rDNase is sensitive to mechanical agitation Dispense into aliquots and store at 20 C The frozen solution is stable for at least 6 months Do not freeze thaw the aliquots more than three times NucleoSpin miRNA Plasma 10 preps 50 preps 250 preps REF 740981 10 740981 50 740981 250 Wash 6 mL 25 mL 2 x 50 mL Buffer MW2 Add 24 mL ethanol Add 100 mL ethanol Add 200 mL ethanol Concentrate to each bottle RNAse free 1 vial size C 1 vial size C 5 vials size C rDNase Add 3 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 2014 Rev 03 9 Small RNA and DNA from plasma 4 Safety instructions The following components ofthe NucleoSpin miRNA Plasma kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features need not be labeled with H and P phrases up to 125 mL or 125 g Mindergef hrliche Eigenschaften m s
12. in 3 Transfer supernatant Transfer Transfer the clear supernatant into a new Collection Tube supernatant 2 mL lid 4 Adjust binding conditions Note Addition of carrier for example 2 ug of glycogen or 400 pL 5 ug of LPA linear polyacrylamide might slightly improve isopropanol the miRNA yield but usually is not necessary Poly A has shown only negligible effects and furthermore might interfere Vortex 5 s with photometric miRNA quantification Add 400 uL isopropanol and vortex for 5 s 12 MACHEREY NAGEL 07 2014 Rev 03 NucleoSpin miRNA Plasma Bind RNA and DNA Place a NucleoSpin miRNA Column in a Collection Tube 2 mL and load the sample onto the column Load sample RT Incubate for 2 min at room temperature 18 25 C 2 min Centrifuge for 30 s at 11 000 x g 11 000 x g 30s Discard the flow through and place the column back into the collection tube Repeat step if necessary If more than 300 uL plasma serum was used repeat this step until all sample is loaded onto the column Optional DNA digest Note Co purified DNA might interfere with qPCR quantifi cation of miRNA The following on column digest degrades bound DNA including miRNA genes However if miRNA specific qPCR detection systems are used or cell free plasma DNA is needed intact for further analysis skip the rDNase digest and proceed directly with step 7 EE 700 pL MW2 Add 700 uL Buffer MW2 to the NucleoSpin
13. larger oligonucleotides 20 uL The silica membrane is not completely wetted Only weakly binding very small oligonucleotides like miRNA are eluted efficiently Larger RNA and DNA are more likely to remain bound to the column The eluted miRNA is highly concentrated 30 uL standard The standard elution buffer volume of 30 uL is sufficient to wet the silica membrane completely It results a high total yield of miRNA RNA DNA and simultaneously maximizes the concentration 50 uL Increasing the elution buffer volume will further increase the final yield but consequently will reduce the concentration The gain in yield will usually not compensate for the loss in sensitivity of miRNA detection caused by the dilution of the eluate Furthermore larger RNA and DNA will be eluted more efficiently which might however be interesting for the analysis of circulating DNA 8 MACHEREY NAGEL 07 2014 Rev 03 Small RNA and DNA from plasma 3 Storage conditions and preparation of working solutions Attention Buffers MLP and MW1 contain guanidinium thiocyanate Wear gloves and goggles Storage conditions Store lyophilized RNase free rDNAse at 4 C on arrival stable for at least one year e All other kit components should be stored at room temperature 18 25 C and are stable for at least one year Storage at lower temperatures may cause precipitation of salts Before starting the first NucleoSpin miRNA Plasma procedure prepar
14. lower temperatures lowers Problem Carry over of ethanol or salt MW2 completely Suboptimal performance 7 of RNA in a downstream efficiency of salt removal 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 6 2 Ordering information Product REF Pack of NucleoSpin miRNA Plasma Exosome Precipitation Solution Serum Plasma Exosome Precipitation Solution Urine NucleoSpin miRNA rDNAse Set 1 vial rDNase size D 7 mL Reaction Buffer for rDNase Proteinase K Collection Tubes 2 mL Visit www mn net com for more detailed product information 740981 10 50 250 740398 12 20 60 740399 12 50 250 740971 10 50 250 740963 740506 740600 10 50 250 preps 12 20 60 mL 12 50 250 mL 10 50 250 preps 1 set 100 mg 1000 MACHEREY NAGEL 07 2014 Rev 03 17 Small RNA and DNA from plasma 6 3 Product use restriction warranty NucleoSpin miRNA Plasma 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 GENER
15. mponents 4 1 1 Kit contents 4 1 2 Reagents consumables and equipment to be supplied by user 5 1 3 About this user manual 5 2 Product description 6 2 1 The basic principle 6 2 2 Kit specifications 6 2 3 Amount of starting material 7 2 4 Proteinase K digest 8 2 5 Addition of carrier 8 2 6 Elution procedures 8 3 Storage conditions and preparation of working solutions 9 4 Safety instructions 10 5 Protocol small RNA and DNA purification from plasma or serum 12 6 Appendix 16 6 1 Troubleshooting 16 6 2 Ordering information 17 6 3 Product use restriction warranty 18 MACHEREY NAGEL 07 2014 Rev 03 3 Small RNA and DNA from plasma 1 Components 1 1 Kit contents NucleoSpin miRNA Plasma 10 preps 50 preps 250 preps REF 740981 10 740981 50 740981 250 Lysis Buffer MLP 3 mL 13 mL 75 mL Protein Precipitation Buffer MPP 5 mL 5 mL 25 mL Reaction Buffer for rDNase 7mL 7mL 30 mL rDNase RNase free lyophilized 1 vial 1 vial 5 vials size C size C size C Wash Buffer MW1 10 mL 10 mL 35 mL Wash Buffer MW2 Concentrate 6 mL 25 mL 2x 50 mL RNase free H O 13 mL 13 mL 13 mL NucleoSpin miRNA Columns 10 50 250 green rings Collection Tubes 1 5 mL 10 50 250 Collection Tubes 2 mL 10 50 250 Collection Tubes 2 mL lid 10 50 250 User manual 1 1 1 For preparation of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 07 2014 Rev 03 Small RNA and DNA from plasma 1 2 Reagents co
16. nsumables and equipment to be supplied by user Reagents 96 100 ethanol e isopropanol Consumables RNase free disposable pipette tips Equipment Manual pipettors Vortexer Centrifuge for microcentrifuge tubes Personal protection equipment lab coat gloves goggles 1 3 About this user manual It is strongly recommended that first time users of the NucleoSpin miRNA Plasma kit read the detailed protocol section 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 purification procedure All technical literature is available on the internet at www mn net com MACHEREY NAGEL 07 2014 Rev 03 5 Small RNA and DNA from plasma 2 Product description 2 1 The basic principle The NucleoSpin miRNA Plasma kit offers the unique feature to isolate small RNA and DNA from plasma without the need to resort to the cumbersome phenol chloroform extraction or a time consuming proteinase digest The sample material is denatured in Lysis Buffer MLP The protein is then precipitated by Protein Precipitation Buffer MPP and pelleted by centrifugation After the removal of protein the binding conditions for nucleic acids are adjusted by adding isopropanol The small RNA and DNA are bound to the NucleoSpin miRNA Column Optionally DNA can be removed by an on column rDNas
17. resale of the products in the responsibility of the reseller to inspect and assure the use of the DNA RNA protein purification products of MACHEREY NAGEL for a well defined and specific application MACHEREY NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in house quality control product documentation and marketing material This MACHEREY NAGEL product is shipped with documentation stating 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 out of accident or improper or abnormal use of this product defects in products or 18 MACHEREY NAGEL 07 2014 Rev 03 Small RNA and DNA from plasma 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 SPECI
18. sen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze rDNase rDNase Iyophilized D Warning 317 334 261 272 280 RNase free rDNase lyophilisiert Achtung 304 340 3424311 3014312 302 352 333 313 MLP Guanidinium thiocyanate Warning 302 412 260 273 30 60 EUH031 301 312 330 Guanidiniumthiocyanat Achtung 30 60 MW1 Guanidinium thiocyanate Danger 225 210 233 1 15 ethanol 55 75 403 235 Guanidiniumthiocyanat 1 15 Gefahr Ethanol 55 75 Hazard phrases H 225 Highly flammable liquid and vapour Fl ssigkeit und Dampf leicht entz ndbar H 302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken H 317 May cause an allergic skin reaction Kann allergische Hautreaktionen verursachen H 334 May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursa chen H 412 Harmful to aquatic life with long lasting effects Sch dlich f r Wasserorganismen mit langfristiger Wirkung EUH 031 Contact with acids liberates toxic gas Entwickelt bei Ber hrung mit S ure giftige Gase 10 MACHEREY NAGEL 07 2014 Rev 03 Small RNA and DNA from plasma Precaution phrases P 210 P 233 P 260 P 261 P 272 P 273 P 280 P 301 312 P 3
19. specific priming mentioned before MACHEREY NAGEL 07 2014 Rev 03 7 Small RNA and DNA from plasma 2 3 1 Preparation of plasma from human EDTA blood Centrifuge fresh blood sample for 10 min at 2 000 x 9 Remove the plasma without disturbing sedimented cells Freeze plasma at 20 C for storage upon DNA isolation Thaw frozen plasma samples prior to DNA isolation and centrifuge for 3 min at 211 000 x g in order to remove residual cells cell debris and particulate matter Use the supernatant for DNA isolation PBOoOD 2 4 Proteinase K digest A short protein digestion step might increase miRNA yield especially for low quality hemolyzed plasma Add 10 uL of Proteinase K 30 ug uL in Buffer PB see ordering information to 300 uL plasma incubate for 10 min at 37 C and then proceed with addition of Buffer MLP according to the protocol 2 5 Addition of carrier To improve RNA DNA binding to the NucleoSpin miRNA Column carrier can be added to the sample after the removal of precipitated protein Slightly higher yields could be found with 2 ug of glycogen or 5 ug of LPA linear polyacrylamide However negligible effects were observed for Poly A which furthermore interferes with a photometric quantification of the purified nucleic acids 2 6 Elution procedures The elution buffer volume does not only influence total yield and concentration of RNA and DNA but does also influence the ratio between very small and
20. uct claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the 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 mn net com Trademarks Disclaimer NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH amp Co KG TaqMan is a registered trademark of Roche Molecular Systems Inc All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency or operation MACHEREY NAGEL 07 2014 Rev 03 19
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