NucleoSpin® RNA Blood - MACHEREY
Contents
1. The use of blood collected in common blood collection tubes with anticoagulant typically EDTA is recommended For frozen blood samples see section 2 3 Check if 7096 ethanol is available to adjust binding conditions For centrifugation a centrifuge with a swing out rotor and appropriate buckets capable of reaching 4 500 x g is required Refer to section 2 2 if less than 1 3 m L whole blood is used Lyse blood Provide 1 3 mL whole blood in a 15 mL tube provided Add 1 3 mL Lysis Buffer DL to the tube and close the lid Mix If necessary shortly spin to clean the lid Add 33 pL Liquid Proteinase K and close the lid Incubate 3 15 min at room temperature 18 25 C vigorously shaking the tube Centrifuge briefly to clean the lid 1 s at 2 000 x g Short spin only Adjust RNA binding conditions Add 1 3 mL 70 ethanol to the tube and mix vigorously Note It is important to thoroughly mix the ethanol into the lysate Recommended Vigorously shake for 5 s e g on a vortexer at medium speed Alternatively pipette the solution up and down 5 times If necessary centrifuge briefly to clean lid 1 s at 2 000 x g Short spin only This centrifugation step can be omitted if the lid is not wetted by the Iysate For example mix by vortexing at medium speed or by pipetting up and down 1 3 mL blood 1 3 mL DL 33 uL Liquid Proteinase K RT 3 15 min 2 000 x g 1s 1 3 mL 70 eth2. r 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 RNA from blood 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 Product description 7 2 1 The basic principle 7 2 2 Kit specifications 8 2 3 Handling preparation and storage of starting materials 9 2 4 Elution procedures 10 3 Storage conditions and preparation of working solutions 11 4 Safety instructions 13 5 NucleoSpin RNA Blood protocols 15 5 1 RNA isolation from 200 uL blood 15 5 2 RNA isolation from 400 uL blood 18 6 NucleoSpin RNA Blood Midi protocol RNA isolation from 1 3 mL blood 21 7 Appendix 24 7 1 rDNase digestion in solution 24 7 2 Troubleshooting 26 7 3 Ordering information 29 7 4 Product use restriction warranty 30 MACHEREY NAGEL 05 2014 Rev 03 3 RNA from blood 1 Components 1 1 Kit contents NucleoSpin RNA Blood 10 preps 50 preps REF 740200 10 740200 50 Lysis Buffer DL 25 mL 25 mL Wash Buffer RB2 13 mL 13 mL Wash Buffer RB3 Concentrate 6 mL 12 mL Membrane Desalting Buffer MDB 10 mL 25 mL Reaction Buffer for rDNase 7 mL 7 mL rDNase RNase free lyophilized 1 vial size C 2 vials size D Liquid Proteinase K 120 uL 600 uL RNase free H O 13 mL 13 mL NucleoSpin RNA Blood Columns 10 50 light blue rings plus Collection Tubes Collection Tubes
3. Ethanol 5 20 Hazard phrases H 226 Flammable liquid and vapour Fl ssigkeit und Dampf 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 verursachen 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 MACHEREY NAGEL 05 2014 Rev 03 13 RNA from blood Precaution phrases P 261 P 280 P 3014312 P 3024352 P 304 340 P 333 313 P 3424311 P 363 Avoid breathing dust Einatmen von Staub 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 comfort able for breathing Bei Einatmen An die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert If skin irritation occurs Get medical advice attention Be
4. RNA from blood User manual NucleoSpin RNA Blood NucleoSpin RNA Blood Midi May 2014 Rev 03 MACHEREY NAGEL MN www mn net com RNA from blood Protocol at a glance Rev 03 NucleoSpin NucleoSpin NucleoSpin RNA Blood RNA Blood RNA Blood Midi 200 uL blood 400 pL blood 1 3 mL blood 1 Lyse blood 200 pL blood 400 uL blood 1 3 mL blood 200 uL DL 400 uL DL 1 8 mL DL Mix Mix Mix 5 uL Pro K 10 uL Pro K 33 uL Pro K RT 3 15 min RT 3 15 min RT 3 15 min shaking shaking shaking 2 Adjust RNA binding 200 pL 70 ethanol 400 uL 70 ethanol 1 3 mL 7096 ethanol conditions Mix Mix Mix 3 Bind RNA Load sample Y Load sample stepwise Load sample 11 000 x g 30s 3 11 000 x g 30s e 3 4 500 x g 3 min 4 Desalt silica membrane 350 uL MDB 350 uL MDB 1 2 mL MDB 11 000 x g 30 s Cc 3 11 000 x g 30s e gt 4 500 x g 3 min 5 Digest DNA br 95 uL rDNase 95 uL rDNase e 240 uL rDNase RT 15 min RT 15 min RT 15 min 6 Wash silica 200 uL RB2 200 uL RB2 m 1 mL RB2 membrane 600 uL RB3 600 uL RB3 n 3 mL RB3 250 uL RB3 250 uL RB3 y T and2 wash 25 11 000xg 30s 11 000xg 30s 4 500x g 3 min 3 wash ES 11 000xg 2min S 11 000 x g 2 min 7 Elute RNA 60 uL RNase 60 uL RNase 200 uL RNase free H O free H O free H O 3 11 000 xg 30s 3 11 000 x g 30 s 3 4 500 x g 3 min MACHEREY NAGEL GmbH amp Co KG Neumann Neander S
5. It is strongly recommended to read the detailed protocol sections of this user manual if using the NucleoSpin RNA Blood or NucleoSpin RNA Blood Midi kits for the first time However experienced users may refer to the Protocol at a glance 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 6 MACHEREY NAGEL 05 2014 Rev 03 RNA from blood 2 Product description 2 1 The basic principle The NucleoSpin RNA Blood kits offer a direct total blood lysis from 200 400 uL NucleoSpin RNA Blood or 400 1300 uL NucleoSpin RNA Blood Midi whole blood collected in standard e g EDTA blood collection tubes One of the most important aspects in RNA purification is to prevent RNA degradation during the isolation With the NucleoSpin RNA Blood method leukocytes the main source of RNA in whole blood and other blood cells are lysed by incubating the whole blood in a solution containing large amounts of chaotropic ions This lysis buffer immediately inactivates RNases which are present in virtually all biological materials and creates appropriate binding conditions that favor adsorption of RNA to the silica membrane A complex selective erythrocyte lysis and preparation of a leukocyte pellet is not necessary Contaminating DNA which is also bound to the silica membrane is r
6. NucleoSpin miRNA NucleoSpin RNAXS rDNase Set Collection Tubes 2 mL Visit www mn net com for more detailed product information 740944 740955 10 20 50 250 740962 20 740933 10 50 250 740966 10 50 250 740971 10 50 250 740902 10 50 250 740963 740600 Suitable for 100 preps 10 20 50 250 20 10 50 250 10 50 250 10 50 250 10 50 250 1 set 1000 DISTRIBUTION AND USE OF NUCLEOSPIN RNA DNA BUFFER SET AND NUCLEOSPIN TRIPREP IN THE USA IS PROHIBITED FOR PATENT REASONS MACHEREY NAGEL 05 2014 Rev 03 29 RNA from blood 7 4 Product use restriction warranty NucleoSpin RNA Blood kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly described in original MACHEREY NAGEL product leaflets MACHEREY NAGEL products are intended for GENERAL LABORATORY USE ONLY MACHEREY NAGEL products are suited for QUALIFIED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet of the product MACHEREY NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products in other fields of application Application on the human body is STRICTLY FORBIDDEN Th
7. 100 mL ethanol Concentrate rDNase 1 vial size C 2 vials size D 2 vials size D RNase free Add 1 mL Reaction Add 2 5 mL Add 2 5 mL lyophilized Buffer for rDNase Reaction Buffer for Reaction Buffer for rDNase to each vial rDNase to each vial 12 MACHEREY NAGEL 05 2014 Rev 03 RNA from blood 4 Safety instructions The following components of the NucleoSpin RNA Blood and NucleoSpin RNA Blood Midi 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 Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze rDNase rDNase lyophilized Warning 317 334 261 280 RNase free rDNase lyophilisiert Achtung 3024352 3044340 3334313 3424311 363 DL Guanidinium thiocyanate Warning 302 412 260 273 30 60 EUHOS1 301 4312 330 Guanidiniumthiocyanat Achtung 30 60 RB2 Guanidine hydrochlo Warning 226 302 210 233 ride 24 36 ethanol 301 312 330 20 35 403 235 Guanidinhydrochlorid Achtung 24 36 Ethanol 20 35 MDB Guanidinium thiocya Warning 226 210 233 nate 1 15 ethanol 403 235 5 20 Guanidiniumthiocyanat Achtung 1 15
8. 2 mL with lid 10 50 for Iysis Collection Tubes 1 5 mL for elution 10 50 Collection Tubes 2 mL 30 150 User manual 1 1 Patent pending For preparation of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 05 2014 Rev 03 RNA from blood 1 1 Kit contents continued NucleoSpin RNA Blood Midi 20 preps REF 740210 20 Lysis Buffer DL 50 mL Wash Buffer RB2 13 mL Wash Buffer RB3 Concentrate 25 mL Membrane Desalting Buffer MDB 50 mL Reaction Buffer for rDNase 7mL rDNase RNase free lyophilized 2 vials size D Liquid Proteinase K 800 uL RNase free H O 13 mL NucleoSpin RNA Blood Midi 20 Columns plus Collection Tubes Collection Tubes 15 mL 60 for Iysis elution and washing steps User manual 1 Patent pending For preparation of working solutions and storage conditions see section 3 MACHEREY NAGEL 05 2014 Rev 03 5 RNA from blood 1 2 Reagents consumables and equipment to be supplied by user Reagents 96 100 ethanol to prepare Wash Buffer RB3 70 ethanol to adjust RNA binding conditions Consumables Sterile RNase free tips Equipment Manual pipettors Vortex mixer Centrifuge for microcentrifuge tubes NucleoSpin RNA Blood Centrifuge for 15 mL tubes with a swing out rotor capable of reaching 4 500 x g NucleoSpin RNA Blood Midi Personal protection equipment e g lab coat gloves goggles 1 3 About this user manual
9. 3 15 min at room temperature 18 25 C vigorously shaking the tube on a shaker e g Eppendorf Thermoshake 1 400 rpm Centrifuge briefly to clean the lid 1 s at 2 000 x g Short spin only Adjust RNA binding conditions Add 400 pL 70 ethanol to the tube and mix vigorously Note It is important to thoroughly mix the ethanol with the lysate Recommended Place tubes in a rack with lid Close the rack lid and and strongly shake the assembly Alternatively pipette the solution up and down 5 times Centrifuge briefly to clean the lid 1 s at 2 000 x g Short spin only b e 400 uL blood 400 pL DL 10 uL Liquid Proteinase K RT 3 15 min 2 000 x g 1s 400 uL 7096 ethanol Mix 2 000 x g 1s 18 MACHEREY NAGEL 05 2014 Rev 03 NucleoSpin RNA Blood isolation from 400 uL blood Bind RNA Transfer 610 uL lysate into a NucleoSpin RNA Blood Column light blue ring placed in a Collection Tube Load 610 uL lysate Note Do not pipette more than 650 uL into the spin column this will cause the column to overflow Avoid foam and aerosol formation Avoid wetting the rim edge of the column Centrifuge 30 s at 11 000 x g and discard flow through and Collection Tube Place the column in a new Collection Tube 2 mL provided 11 000 x g c 30s Apply the remaining lysate into the NucleoSpin RNA Blood Column Load residual Note Do not pi
10. Kit specifications NucleoSpin RNA Blood kits are recommended for the isolation of RNA from whole blood e g stabilized with EDTA citrate or heparin The NucleoSpin RNA Blood kits allow the purification of RNA with an A eo Ago ratio typically exceeding 1 9 measured in TE buffer pH 7 5 The isolated RNA is ready to use for typical downstream applications e g reverse transcriptase PCR RT PCR RNA isolated with the NucleoSpin RNA Blood kits is typically of high integrity However RNA integrity strongly depends on the sample quality The amount of DNA contamination is significantly reduced during on column digestion with rDNase However in very sensitive applications it may be possible to detect traces of DNA The probability of DNA detection with PCR increases with 1 the number of DNA copies per preparation single copy target plastidial mitochondrial target plasmid transfected into cells 2 decreasing PCR amplicon size Table 1 Kit specifications at a glance Parameter NucleoSpin RNA Blood NucleoSpin RNA Blood Midi Sample material 200 400 pL fresh or frozen 400 1300 uL fresh or frozen whole blood e g stabilized whole blood e g stabilized with EDTA citrate or heparin with EDTA citrate or heparin Format Mini spin column Midi spin column Fragment size gt 200 nt gt 200 nt Typical yield 7 ug 3 20 ug per 1 mL 7 ug 3 20 ug per 1 mL blood from healthy subjects blood
11. PCR targets e g gt 500 bp or intron spanning primers if possible Use protocol 7 1 for subsequent rDNase digestion in so lution Carry over of ethanol or salt Do not let the flow through touch the column outlet after the second Buffer RB3 wash Be sure to centrifuge at the corresponding speed for the respective time in order to remove ethanolic Buffer RB3 completely Suboptimal Check if Buffer RB3 has been equilibrated to room performance temperature before use Washing at lower temperatures of RNA in lowers efficiency of salt removal by Buffer RB3 downstream experiments 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 28 MACHEREY NAGEL 05 2014 Rev 03 RNA from blood 7 3 Ordering information Product REF Pack of NucleoSpin RNA Blood 740200 10 50 10 50 NucleoSpin RNA Blood Midi 740210 20 20 NucleoSpin 8 RNA Blood 740220 5 12x8 60x8 NucleoSpin 96 RNA Blood 740225 2 4 2x96 4x96 NucleoSpin miRNA Plasma 740981 10 50 250 10 50 250 NucleoSpin RNA Clean up 740948 10 50 250 10 50 250 NucleoSpin RNA Clean up XS 740903 10 50 250 10 50 250 NucleoSpin RNA DNA Buffer Set NucleoSpin RNA NucleoSpin RNA Midi NucleoSpin RNA Protein NucleoSpin TriPrep
12. from healthy subjects Aaco Acso 1 9 2 1 1 9 2 1 Elution volume 40 120 uL 200 400 uL Binding capacity 200 ug 700 ug Preparation time 45 min 6 preps excl lysis 85 min 6 preps excl lysis 8 MACHEREY NAGEL 05 2014 Rev 03 RNA from blood The NucleoSpin RNA Blood kit contains one protocol that allows the use of 200 uL of whole blood by a total direct blood lysis and a second protocol for processing 400 uL of whole blood with a second loading step The NucleoSpin RNA Blood Midi kit contains a protocol that allows 1 3 mL of whole blood by a total direct blood lysis If other volumes than 200 pL 400 uL or 1300 uL blood are used adjust the volumes of Buffer DL and 70 ethanol in step 1 and 2 of the corresponding protocol by maintaining the following ratio 1 1 1 sample Buffer DL 70 96 ethanol Example 300 uL blood 300 uL Buffer DL 300 uL 70 ethanol The volume of Proteinase K can be calculated as follows Blood volume pL 40 volume Proteinase K uL Example 300 uL blood 40 7 5 uL Liquid Proteinase K The isolated RNA can be used as a template in RT PCR reactions Generally 1 40 96 of the eluate from RNA prepared with 200 400 uL blood is suitable as a template for RT PCR If possible intron spanning primers should be used for RT PCR 2 3 Handling preparation and storage of starting materials NucleoSpin RNA Blood kits are designed for isolation of total RNA from fresh human whole blood Whole blood
13. anol Mix 2 000 x g 1s MACHEREY NAGEL 05 2014 Rev 03 21 NucleoSpin RNA Blood Midi isolation from 1 3 mL blood 3 Bind RNA m Transfer the complete lysate 4000 pL into a Load NucleoSpin RNA Blood Midi Column placed in a 15 mL pac max Collection Tube 4000 pL lysate Do not pipette more than 4000 uL into the Midi column this will cause the column to overflow Avoid foam and aerosol formation Centrifuge 3 min at 4 500 x g and leave the column in the 4 500 x g tube with the flow through 3 min 4 Desalt silica membrane T Add 1 2 mL MDB Membrane Desalting Buffer onto 1 2 mL MDB the column and centrifuge 3 min at 4 500 x g Discard flow through and Collection Tube and place the V column in a new Collection Tube 15 mL provided e gt 4 500 x g 3 min 5 Digest DNA 240 uL rDNase Add 240 uL rDNase onto the column Incubate at room temperature for 15 min centrifugation 1 i after this incubation is not necessary m n 6 Wash and dry silica membrane T 1 wash 1 mL RB2 Add 1 mL Buffer RB2 to the NucleoSpin RNA Blood Le Midi Column Centrifuge for 3 min at 4 500 x g V Leave the NucleoSpin RNA Blood Midi Column in the tube c 4 500 x 9 with the flow through 3 min 22 MACHEREY NAGEL 05 2014 Rev 03 NucleoSpin RNA Blood Midi isolation from 1 3 mL blood Add 3 mL Buffer RB3 to the NucleoSpin RNA Blood Midi Column Centrifuge for 3 min at 4 500 x g Place t
14. ase according to instructions given in section 3 Store other kit components at room temperature Storage at low temperatures may cause salt precipitation Keep bottles tightly closed in order to prevent evaporation or contamination lonic strength and pH influence Azs absorption as well as ratio Azeo A280 For adsorption measurement use 5 mM Tris pH 8 5 as diluent Please see also Manchester K L 1995 Value of A eo zgo ratios for measurement 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 26 MACHEREY NAGEL 05 2014 Rev 03 RNA from blood Problem Possible cause and suggestions Sample material Bad sample quality Make sure blood is collected into a standard blood collection tube e g EDTA tube according to the manufacturer s instructions using fresh blood is always Cl d recommended Sample should be stored at 4 C for no longer 099 than 24 hours Freeze sample if it is not possible to process NucleoSpin within one day Column P RNA Gal Inappropriate lysis binding conditions or yield Do not premix Liquid Proteinase K with Lysis Buffer DL Contamination of RNA with genomic DNA Make sure to vigorously shake during lysis incubation shaking is essential for the procedure Make sure to use 70 etha
15. bed in the individual protocols recovery rate about 70 90 there are several modifications possible High yield Perform two elution steps with the volume indicated in the individual protocol About 90 100 96 of bound nucleic acids will be eluted High yield and high concentration Elute with the standard elution volume and apply the eluate once more onto the column for re elution Eluted RNA should be immediately placed and kept on ice for optimal stability and to prohibit omnipresent RNases general lab ware fingerprints dust from degrading the RNA For short term storage freeze at 20 C for long term storage freeze at 70 C 10 MACHEREY NAGEL 05 2014 Rev 03 RNA from blood 3 Storage conditions and preparation of working solutions Attention Buffers DL RB2 and MDB contain chaotropic salts Wear gloves and goggles CAUTION Buffers DL RB2 and MDB contain guanidinium salts which can form highly reactive compounds when combined with bleach sodium hypochlorite DO NOT add bleach or acidic solutions directly to the sample preparation waste Store lyophilized rDNase RNase free at 4 C on arrival stable up to 1 year Lysis Buffer DL is light sensitive during long time storage Therefore Lysis Buffer DL is provided in a black bottle Short light exposure several hours does not affect the buffer After first use it is recommended to store Liquid Proteinase K at 4 C or 20 C All other kit compone
16. e respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for N VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for N VITRO diagnostic use Please pay attention to the package of the product N VITRO diagnostic products are expressly marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR N VITRO DIAGNOSTIC USE ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE No claim or representations is intended for its use to identify any specific organism or for clinical use included but not limited to diagnostic prognostic therapeutic or blood banking It is rather in the responsibility of the user or in any case of resale of the products in the responsibility of the reseller to inspect and assure the use of the DNA RNA protein purification products of MACHEREY NAGEL for a well defined and specific application MACHEREY NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in house quality control product documentation and marketing material This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrant
17. emoved by a recombinant DNase solution supplied which is directly applied onto the silica membrane during the preparation Simple washing steps with two different buffers remove salts metabolites and macromolecular cellular components Pure RNA is finally eluted under low ionic strength conditions with RNase free H O supplied The RNA preparation using NucleoSpin RNA Blood kits is performed at room temperature A refrigerated centrifuge is not necessary The eluate however should be handled with care because 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 Simultaneous isolation of RNA and DNA NucleoSpin RNA DNA Buffer Set The NucleoSpin RNA DNA Buffer Set see ordering information is a support set for RNA and DNA isolation in conjunction with NucleoSpin RNA NucleoSpin RNA XS NucleoSpin miRNA NucleoSpin RNA Plant NucleoSpin RNA Protein and NucleoSpin RNA Blood This patented technology enables successive elution of DNA and RNA from one NucleoSpin Column with low salt buffer and water respectively DNA and RNA are immediately ready for downstream applications DISTRIBUTION AND USE OF NUCLEOSPIN RNA DNA BUFFER SET AND NUCLEOSPIN TRIPREP IN THE USA IS PROHIBITED FOR PATENT REASONS MACHEREY NAGEL 05 2014 Rev 03 7 RNA from blood 2 2
18. esidual buffer from the previous steps is washed away with Buffer RBS especially if the lysate has been in contact with the inner rim of the column during loading of the lysate onto the column For efficient washing of the inner rim flush it with Buffer RB3 Add 250 pL Buffer RB3 to the NucleoSpin RNA Blood Column Centrifuge for 2 min at 11 000 x g In this step ethanol is removed from the column Place the column into a nuclease free Collection Tube 1 5 mL provided and discard the Collection tube with flow through from the previous step If for any reason the liquid level in the Collection Tube has reached the NucleoSpin RNA Blood Column after centrifugation discard flow through and centrifuge again Elute RNA Add 60 uL RNase free H O supplied onto the column and centrifuge 30 s at 11 000 x g The RNA is eluted into the Collection Tube For alternative elution procedures see section 2 4 200 uL RB2 11 000 x g 30s 600 uL RB3 11 000 x g 30s 250 uL RB3 11 000 x g 2 min 60 uL RNase free H O 11 000 x g 30s 20 MACHEREY NAGEL 05 2014 Rev 03 NucleoSpin RNA Blood Midi isolation from 1 3 mL blood NucleoSpin RNA Blood Midi protocol RNA isolation from 1 3 mL blood Before starting the preparation Check if Wash Buffer RB3 and rDNase were prepared according to section 3 The complete procedure should be performed at room temperature 18 25 C
19. g genomic DNA Especially if high copy number targets are analyzed e g multi gene family mitochondrial plastidal or plasmid targets from transfections the target gene is of a very low expression level the amplicon is relatively small 200 bp DNA digestion in solution can efficiently destroy contaminating DNA However stringent RNase control and subsequent re purification of the RNA in order to remove buffer salts rDNase and digested DNA are usually required The high quality recombinant RNase free DNase rDNase in the NucleoSpin RNA Blood kits can also be used for a digestion in solution in order to remove trace amounts of contaminating DNA 1 Digest DNA Reaction setup Add 0 5 pL rDNase per 10 uL eluted RNA and mix moderately Centrifuge briefly 1 s at 2 000 x g to collect all liquid in the lower part of the tube Note This step is important to ensure that every droplet of the RNA comes into contact with the rDNase to ensure efficient DNA digestion 2 Incubate sample Incubate for 10 min at 37 C 24 MACHEREY NAGEL 05 2014 Rev 03 RNA from blood Repurify RNA Repurify RNA with a suitable RNA cleanup procedure for example using the NucleoSpin RNA Clean up kit see ordering information or by ethanol precipitation Ethanol precipitation exemplary Add 0 1 volume of 3 M sodium acetate pH 5 2 and 2 5 volumes of 96 100 ethanol to one volume of sample Mix thorough
20. gh The flow through may be slightly brown The flow through can remain in the tube without disturbing DNA digestion Digest DNA Add 95 uL rDNase onto the column Incubate at room temperature for 15 min Note Centrifugation after incubation is not necessary Wash and dry silica membrane Add 200 uL Buffer RB2 to the NucleoSpin RNA Blood Column Centrifuge for 30 s at 11 000 x g Discard flow through and Collection Tube and place the column into a new Collection Tube 2 mL provided Buffer RB2 will inactivate the rDNase Load lysate 11 000 x g 30s 350 pL MDB 11 000 x g 30s 95 pL rDNase RT 15 min 200 pL RB2 11 000 x g 30s 16 MACHEREY NAGEL 05 2014 Rev 03 NucleoSpin RNA Blood isolation from 200 uL blood Add 600 uL Buffer RB3 to the NucleoSpin RNA Blood Column Centrifuge for 30 s at 11 000 x g Discard flow through and place the column into a new Collection Tube 2 mL provided Note Make sure that residual buffer from the previous steps is washed away with Buffer RB3 especially if the lysate has been in contact with the inner rim of the column during loading of the lysate onto the column For efficient washing of the inner rim flush it with Buffer RB3 Add 250 pL Buffer RB3 to the NucleoSpin RNA Blood Column Centrifuge for 2 min at 11 000 x g In this step ethanol is removed from the column Place the column into a nuclease free Collection Tube 1 5
21. he column into a nuclease free Collection Tube 15 mL provided and discard the Collection Tube with flow through from the previous step In this step the ethanol is removed from the column Make sure that no flow through spills at the column outlet visually inspect that all column outlets are dry Elute RNA Add 200 uL RNase free H O supplied onto the column Centrifuge for 3 min at 4 500 x g The RNA is eluted into the Collection Tube An additional elution step with 200 pL fresh elution buffer will increase the total yield by approximately 25 m lt Q a 3 mL RB3 4 500 x g 3 min 200 uL RNase free H O 4 500 x g 3 min MACHEREY NAGEL 05 2014 Rev 03 23 RNA from blood 7 Appendix 7 1 rDNase digestion in solution The on column rDNase digestion in the standard protocol is already very efficient and results in minimal residual DNA This DNA will not be detectable in most downstream applications Despite this there are still certain applications which require even lower contents of residual DNA However 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 A typical 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 contaminatin
22. i Hautreizung rztlichen Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTER doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM ArzV anrufen Wash contaminated clothing before reuse Kontaminierte Kleidung vor erneutem Waschen tragen For further information please see Material Safety Data Sheets www mn net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenbl ttern www mn net com 14 MACHEREY NAGEL 05 2014 Rev 03 NucleoSpin RNA Blood isolation from 200 uL blood 5 5 1 NucleoSpin RNA Blood protocols RNA isolation from 200 uL blood Before starting the preparation Check if Wash Buffer RB3 and rDNase were prepared according to section 3 The complete procedure should be performed at room temperature 18 25 C The use of blood collected in common blood collection tubes with anticoagulant typically EDTA is recommended For frozen blood samples see section 2 3 Check if 7096 ethanol is available to adjust binding conditions Refer to section 2 2 if less than 200 uL whole blood is used Lyse blood Provide 200 uL whole blood in a Collection Tube 2 mL with lid provided Add 200 uL Lysis Buffer DL to the tube and close the lid Mix If necessary shortly spin to clean the lid Add 5 pL Liquid Proteinase K and close the lid Incubate 3 15 min at room temperature 18 25 C vigorously shaking the tube o
23. ly Incubate several minutes to several hours at 20 C or 4 C Note Choose long incubation times if the sample contains low RNA concentration Short incubation times are sufficient if the sample contains high RNA concentration Centrifuge for 10 min at maximum speed Wash RNA pellet with 7096 ethanol Dry RNA pellet and resuspend RNA in RNase free H O MACHEREY NAGEL 05 2014 Rev 03 25 RNA from blood 7 2 Troubleshooting Problem Possible cause and suggestions RNA is degraded no RNA obtained RNase contamination Create an RNase free working environment Wear gloves during all steps of the procedure Change gloves frequently Use of sterile 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 RNA quality or yield Reagents not applied or restored properly Reagents not properly restored Add the indicated volume of Reaction Buffer for rDNase and 96 ethanol to Buffer RB3 Concentrate and mix Reconstitute and store lyophilized rDNase 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 Kit storage Reconstitute and store lyophilized rDN
24. mL supplied and discard the Collection tube with flow through from the previous step If for any reason the liquid level in the Collection Tube has reached the NucleoSpin RNA Blood Column after centrifugation discard flow through and centrifuge again Elute RNA Add 60 uL RNase free H O supplied onto the column and centrifuge 30 s at 11 000 x g The RNA is eluted into the Collection Tube For alternative elution procedures see section 2 4 600 pL RB3 11 000 x g 30s 250 uL RB3 11 000 x g 2 min 60 uL RNase free H O 11 000 x g 30s MACHEREY NAGEL 05 2014 Rev 03 17 NucleoSpin RNA Blood isolation from 400 uL blood 5 2 RNA isolation from 400 pL blood Before starting the preparation Check if Wash Buffer RB3 and rDNase were prepared according to section 3 The complete procedure should be performed at room temperature 18 25 C The use of blood collected in common blood collection tubes with anticoagulant typically EDTA is recommended For frozen blood samples see section 2 3 Check if 7096 ethanol is available to adjust binding conditions Refer to section 2 2 if less than 400 uL whole blood is used Lyse blood Provide 400 uL whole blood in a Collection Tube 2 mL with lid provided Add 400 uL Lysis Buffer DL to the tube and close the lid Mix If necessary shortly spin to clean the lid Add 10 pL Liquid Proteinase K and close the lid Incubate
25. n a shaker e g Eppendorf Thermoshaker 1 400 rpm Centrifuge briefly to clean the lid 1 s at 2 000 x g Short spin only Adjust RNA binding conditions Add 200 pL 70 ethanol to the tube and mix vigorously Note It is important to thoroughly mix the ethanol with 200 uL blood 200 pL DL 5 pL Liquid Proteinase K RT 3 15 min 2 000 x g 1s 200 uL 7096 ethanol the lysate Recommended Place tubes in a rack with lid Mix Close the rack lid and and strongly shake the assembly Alternatively pipette the solution up and down 5 times Centrifuge briefly to clean the lid 1 s at 2 000 x g e 2 000 x g Short spin only is MACHEREY NAGEL 05 2014 Rev 03 15 NucleoSpin RNA Blood isolation from 200 uL blood Bind RNA Adjust pipette to 610 uL and transfer lysate into a NucleoSpin RNA Blood Column placed in a Collection Tube Note Do not pipette more than 650 uL into the spin column this will cause the column to overflow Avoid formation of foam and aerosols Avoid wetting the rim edge of the column Centrifuge 30 s at 11 000 x g Discard flow through and Collection Tube Place the column in a new Collection Tube 2 mL provided Desalt silica membrane Add 350 uL MDB Membrane Desalting Buffer onto the column and centrifuge 30 s at 11 000 x g Note After centrifugation the column can remain in the Collection Tube including the flow throu
26. nol in this procedure to adjust binding conditions rDNase not active e Reconstitute and store lyophilized rDNase according to instructions given in section 3 DNase solution not properly applied Pipette rDNase solution directly onto the center of the silica membrane High leukocyte number The higher the leukocyte number the higher the risk to detect residual DNA in the eluted RNA To avoid this use less blood DNA detection system too sensitive The amount of DNA contamination is effectively reduced during the on column digestion with rDNase However it cannot be guaranteed that the purified RNA is 10096 free of DNA Therefore in very sensitive applications it might still be possible to detect DNA The NucleoSpin RNA system was checked by the following procedure One million HeLa cells are subjected to RNA isolation RNA eluate is used as a template for PCR detection of a 1 kb fragment in a 30 cycle reaction Generally no PCR product is obtained while skipping the DNase digest usually leads to positive PCR results MACHEREY NAGEL 05 2014 Rev 03 27 RNA from blood Problem Possible cause and suggestions Contamination The probability of DNA detection with PCR increases with the number of DNA copies per preparation single copy target plastidial mitochondrial target plasmid transfected into cells of RNA with decreasing of PCR amplicon size genomic DNA continued Use larger
27. nts should be stored at room temperature 18 25 C and are stable up to one year Storage at lower temperatures may cause precipitation of salts Check that 7096 ethanol is available as additional solution to adjust RNA binding conditions Check that 96 100 ethanol is available as additional solution to prepare Wash Buffer RB3 Before starting any NucleoSpin RNA Blood protocol prepare the following rDNase RNase free Add indicated volume of Reaction Buffer for rDNase see table below to the rDNase vial and incubate for 1 min at room temperature Gently swirl the vials 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 the aliquots more than three times Be careful when opening the vial as some particles of the lyophilisate may be attached to the lid Wash Buffer RB3 Add the indicated volume of 96 100 96 ethanol see table below to Buffer RB3 Concentrate Mark the label of the bottle to indicate that ethanol was added Store Wash Buffer RB3 at room temperature 18 25 C for up to one year MACHEREY NAGEL 05 2014 Rev 03 11 RNA from blood NucleoSpin NucleoSpin RNA Blood RNA Blood Midi 10 preps 50 preps 20 preps REF 740200 10 740200 50 740210 20 Wash 6 mL 12 mL 25 mL Buffer RB3 Add 24 mL ethanol Add 48 mL ethanol Add
28. pette more than 650 uL into the spin column lysate this will cause the column to overflow Avoid foam and aerosol formation Avoid wetting the rim edge of the column Centrifuge 30 s at 11 000 x g Discard flow through and Collection Tube Place the column in a new Collection Tube 2 mL provided 11 000 x g c 30s Desalt silica membrane Add 350 uL MDB Membrane Desalting Buffer onto the f L MDB column and centrifuge 30 s at 11 000 x g EODEM Note After centrifugation the column can remain in the 11 000 x g Collection Tube including the flow through The flow through es 30s might be slightly brown The flow through can remain in the tube without disturbing DNA digestion Digest DNA 95 pL Add 95 pL rDNase onto the column Incubate at room rDNase temperature for 15 min RT 15 min Note Centrifugation after incubation is not necessary MACHEREY NAGEL 05 2014 Rev 03 19 NucleoSpin RNA Blood isolation from 400 uL blood Wash and dry silica membrane Add 200 uL Buffer RB2 to the NucleoSpin RNA Blood Column Centrifuge for 30 s at 11 000 x g Discard flow through and Collection Tube Place the column into a new Collection Tube 2 mL provided Buffer RB2 will inactivate the rDNase Add 600 pL Buffer RB3 to the NucleoSpin RNA Blood Column Centrifuge for 30 s at 11 000 x g Discard flow through and place the column back into the Collection Tube Note Make sure that the r
29. r 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 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 con
30. s 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 30 MACHEREY NAGEL 05 2014 Rev 03 RNA from blood There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whethe
31. should be collected in the presence of an anticoagulant preferably EDTA citrate or heparin It is highly recommended to process blood samples within a few hours after collecting them when EDTA citrate or heparin collection tubes are used Samples should be stored at 4 C for no longer than 24 hours The mRNAs contained in blood cells have different stabilities As a result in order to ensure that the isolated RNA contains a representative distribution of mRNAs blood samples should not be stored for long periods before isolating RNA If frozen blood samples have to be processed aliquots of 200 uL 400 uL or 1300 uL preferably of frozen blood aliquots should be quickly thawed in the presence of 1 volume Lysis Buffer DL while shaking If long term storage of stabilized whole blood is necessary it is recommended storing the lysates at 20 C For this add the indicated volume of Lysis Buffer DL to the blood sample without adding Liquid Proteinase K Store the lysates at 20 C After thawing add Liquid Proteinase K and follow the protocol at step 1 Wear gloves at all times during the preparation Change gloves frequently MACHEREY NAGEL 05 2014 Rev 03 9 RNA from blood 2 4 Elution procedures It is possible to adjust the elution method and volume of RNase free water used for the subsequent application of interest refer to Table 1 regarding suitable ranges of elution volumes In addition to the standard method descri
32. tact MACHEREY NAGEL GmbH amp Co KG Tel 49 24 21 969 270 tech bio 9 mn net com MACHEREY NAGEL 05 2014 Rev 03 31
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