Total RNA Isolation
Contents
1. Total RNA Isolation User Manual NucleoSpin RNA Il NucleoSpin RNA L January 2010 Rev 11 MACHEREY NAGEL MN Total RNA Isolation Protocol at a glance Rev 11 NucleoSpin RNA II NucleoSpin RNA L 1 Homogenize sample 30 mg 100 mg 2 Lyse cells m 350 ul RA1 1 8 ml RA1 3 5 ul B mercaptoethanol 18 ul B mercaptoethanol Mix ji Mix 3 Filtrate lysate j 11 000 x g Y 4 500 x g c 1 min j c 10 min 4 Adjust RNA 350 yl 70 ethanol 1 8 ml 70 ethanol binding j conditions Mix Mix 5 Bind RNA KE Load sample il Load sample 5 11 000 x g 4 500 x g 30s i 3 min V V 6 Desalt silica membrane 350 ul MDB 2 2 ml MDB j 11 000 x g c 4 500 x g 1 min D 3 min V V 7 Digest DNA m j 95 yl DNase L 250 ul DNase E reaction mixture Y reaction mixture RT 15 min RT 15 min id V 8 Wash and dry P Millen membane SS 1 wash 200 jil RA2 i i wash 2 6 ml RA2 E 25 wash 600 ul RAS 2 wash 2 6 ml RA3 6 i Ww 3 wash 250 yl RA3 V 3 wash 2 6 ml RA3 11 000 x g 4st and 2nd 4 500 x g st nd tana CD 30s 3 min 11 000 x g gn 4 500xg rd 2 c5 2 min 5 min 9 Elute highly 500 ul RNase pure RNA SF 60 pl vd free H O free H eS i RT 2 min t 11 000 x g 1 min 4 500 x g 3 min Tel 49 0 24 21 969 270 www mn net com e mail tech bio mn net com MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 D 52355 D ren2. 01 2010 Rev 11 Total RNA Isolation 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions RNase contamination RNA is e Create an RNase free working environment Wear gloves degraded during all steps of the procedure Change gloves frequently no RNA Use of sterile disposable polypropylene tubes is recom ebisined mended Keep tubes closed whenever possible during the preparation Glassware should be oven baked for at least 2 hours at 250 C before use Reagents not applied or restored properly e Reagents not properly restored Add the indicated volume of RNase free H O to rDNase vial and 96 ethanol to Buffer RA3 Concentrate and mix Reconstitute and store lyophilized rDNase according to instructions given in section 3 e Sample and reagents have not been mixed completely Always vortex vigorously after each reagent has been added e No ethanol has been added after lysis Binding of RNA to the silica membrane is only effective in the presence of ethanol Kit storage e Reconstitute and store lyophilized rDNase according to Poor RNA instructions given in section 3 quality or yield e Store other kit components at room temperature Storage at low temperatures may cause salt precipitation e Keep bottles tightly closed in order to prevent evaporation or contamination lonic strength and pH influence A absorption as well as ratio A od oso e For adsorption measurement use 5 mM Tris pH 8 5 as dilu
3. 2 ml apply the mixture and centrifuge for 1 min at 11 000 x g The lysate may be passed alternatively 5 times through a 0 9 mm needle 20 gauge fitted to a syringe In case of visible pellet formation depending on sample amount and nature transfer supernatant without any formed pellet to a new 1 5 ml microcentrifuge tube not supplied Important To process higher amounts of cells gt 1 x 109 or tissue gt 10 mg the lysate should first be homo genized using the 0 9 mm needle 20 gauge followed by filtration through NucleoSpin Filters Adjust RNA binding conditions Discard the NucleoSpin Filter and add 350 pl ethanol 70 to the homogenized lysate and mix by pipetting up and down 5 times Alternatively transfer flow through into a new 1 5 ml microcentrifuge tube not provided add 350 pl ethanol 70 and mix by vortexing 2 x 5 s After addition of ethanol a stringy precipitate may become visible which will not affect the RNA isolation Be sure to disaggregate any precipitate by mixing and load all of the precipitate on the column as described in step 5 Do not cen trifuge the ethanolic lysate before loading it onto the column in order to avoid pelleting the precipitate Bind RNA For each preparation take one NucleoSpin RNA II Column light blue ring placed in a Collection Tube Pipette lysate up and down 2 3 times and load the lysate to the column Centrifuge for 30 s at 11 000 x g Place th
4. BisTris Bis 2 hydroxyethyl imino tris hydroxymethyl methane Sorbitol and lyticase or zymolase for homogenization by enzymatic digestion or a swing mill and glass beads for homogenization by mechanical disruption Before starting the preparation Check if Wash Buffer RA3 and rDNase were prepared according to section 3 Homogenize sample Two alternative protocols are given for homogenization of yeast cells Users may choose between an enzymatic digestion A or mechanical homogenization B depending on laboratory equipment and personal preference Homogenization by enzymatic digest is only recommended for fresh harvested cells homogeni zation by mechanical disruption may also be performed with yeast cell pellets stored at 70 C for several months Note Due to the much higher concentration of genome equivalents in a nucleic acid preparation of yeasts compared with cultured cells or tissue material it may be nec essary to use a lower quantity of cells for the preparation A Homogenization by enzymatic digest Harvest an appropriate amount of cells from YPD culture 5 000 x g 10 min Resuspend pellet in an appropriate amount of fresh prepared sorbitol lyticase buffer 50 100 U lyticase or zymolase in 1 M sorbitol 100 mM EDTA and incubate at 30 C for 30 min Pellet the resulting spheroplasts by centrifugation 1 000 x g 10 min Carefully discard suprnatant It may be necessary to optimize incubation time and lytic
5. Buffer 7ml for rDNase rDNase RNase free lyophilized 1 vial size D RNase free H O 15 ml NucleoSpin Filters L 20 plus Collection Tubes NucleoSpin RNA L Columns 20 plus Collection Tubes Collection Tubes 15 ml 20 User Manual 1 For preparation of working solutions and storage conditions see section 3 6 MACHEREY NAGEL 01 2010 Rev 11 Total RNA Isolation 1 2 Reagents consumables and equipment to be supplied by user Reagents e 96 100 ethanol to prepare Wash Buffer RA3 e 70 ethanol to adjust RNA binding conditions e Reducing agent B mercaptoethanol or DTT dithiothreithol or TCEP BisTris Bis 2 hydroxyethyl imino tris hydroxymethyl methane as supplement for Lysis Buffer RA1 Consumables e 1 5 ml microcentrifuge tubes NucleoSpin RNA II or 15 ml tubes NucleoSpin RNAL e Sterile RNase free tips Equipment Manual pipettors e NucleoSpin RNA II centrifuge for microcentrifuge tubes NucleoSpin RNA L centrifuge for 15 ml tubes with a swing out rotor and appropriate buckets capable of reaching 4 000 4 500 x g e Equipment for sample disruption and homogenization see section 2 3 e Personal protection equipment e g lab coat gloves goggles 1 3 About this User Manual It is strongly recommended reading the detailed protocol sections of this User Manual if the NucleoSpin RNA II or NucleoSpin RNA L kit is used for the first time Experienced users however may
6. Germany Total RNA Isolation Table of contents 1 Components 5 1 1 Kit contents 5 1 2 Reagents consumables and equipment to be supplied by user 7 1 3 About this User Manual 7 2 Product description 8 2 1 The basic principle 8 2 3 Handling preparation and storage of starting materials 13 2 4 Elution procedures 14 3 Storage conditions and preparation of working solutions 15 4 Safety instructions risk and safety phrases 17 5 Protocols 18 5 1 Total RNA purification from cultured cells and tissue with NucleoSpin RNA II 18 5 2 Support protocol NucleoSpin RNA II Total RNA preparation from biological fluids e g serum culture medium 22 5 3 Support protocol NucleoSpin RNA II Total RNA preparation from up to 10 bacterial cells 23 5 4 Support protocol NucleoSpin RNA II Total RNA preparation from up to 5 x 107 yeast cells 25 5 5 Support protocol NucleoSpin RNA II Total RNA preparation from paraffin embedded tissue 27 5 6 Total RNA purification from cultured cells and tissue with NucleoSpin RNA L 28 5 7 Support protocol NucleoSpin RNA L Total RNA preparation from up to 5 x 10 bacterial cells 31 5 8 Support protocol NucleoSpin RNA L Total RNA preparation from up to 3 x 10 yeast cells 32 5 9 Support protocol NucleoSpin RNA II and NucleoSpin RNA L Clean up of RNA from reaction mixtures 34 5 10 Support protocol NucleoSpin RNA II and NucleoSpin RNA L Total RNA preparation from RNA ate treated samples 36 5 11
7. Support protocol NucleoSpin RNA II and NucleoSpin RNA L rDNase digestion in solution 37 MACHEREY NAGEL 01 2010 Rev 11 3 Total RNA Isolation 6 Appendix 6 1 Troubleshooting 6 2 Ordering information 6 3 Product use restriction warranty 39 39 42 43 4 MACHEREY NAGEL 01 2010 Rev 11 Total RNA Isolation 1 Components 1 1 Kit contents NucleoSpin RNA II 10 preps 20 preps 50 preps 250 preps Cat No 740955 10 740955 20 740955 50 740955 250 Lysis Buffer RA1 10 ml 10 ml 25 ml 125 ml Wash Buffer RA2 15 ml 15 ml 15 ml 80 ml Wash Euler RAS 5 ml 5 ml 12 5 ml 3 x 25 ml Concentrate Membrane Desalting Buffer MDB 10 ml 10 ml 25 ml 125 ml Reaction Buffer 3 mi 3 mi 7 ml 35 ml for rDNase rDNase RNase free 1 vial 1 vial 1 vial 5 vials lyophilized size C size C size D size D RNase free H O 5 ml 5 ml 15 ml 65 ml m NucleoSpin Filters 10 20 50 250 violet rings NucleoSpin RNA II Columns light 10 20 50 250 blue rings plus Collection Tubes Collection Tubes 30 60 150 750 2 ml Collection Tubes 10 20 50 250 1 5 ml User Manual 1 1 1 1 For preparation of working solutions and storage conditions see section 3 MACHEREY NAGEL 01 2010 Rev 11 5 Total RNA Isolation 1 4 Kit contents continued NucleoSpin RNA L 20 preps Cat No 740962 20 Lysis Buffer RA1 80 ml Was Buffer RA2 60 ml Wash Buffer RA3 Concentrate 25 ml Membrane Desalting Buffer MDB 50 ml Reaction
8. in 200 pl TE buffer 10 mM Tris HCl 1 mM EDTA pH 8 containing 1 mg ml lysozyme by vigorous vortexing Incubate at 37 C for 10 min For preparation of RNA from Gram positive bacteria resuspend cells in 200 ul TE containing 2 mg ml lysozyme It may be necessary to optimize incubation time and lysozyme concentration depending on the bacterial strain 2 Lysecells Add 1 8 ml Buffer RA1 and 18 pl B mercaptoethanol to the suspension and vortex vigorously For appropriate sample and lysis buffer amounts see section 2 2 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 3 filtrate lysate Reduce viscosity and turbidity of the solution by filtration through NucleoSpin Filters L Apply the lysate to a NucleoSpin Filter L placed in a Collection Tube and centrifuge for 10 min at 4 500 x g In case of visible pellet formation depending on sample amount and nature transfer supernatant without any formed pellet to a new 15 ml centrifuge tube not supplied Proceed with step 4 of the NucleoSpin RNA L standard protocol section 5 6 MACHEREY NAGEL 01 2010 Rev 11 31 NucleoSpin RNA L 5 8 Support protocol NucleoSpin RNA L Total RNA preparation from up to 3 x 10 yeast cells Additional reagents and components to be supplied by user Reducing agent B mercaptoethanol or DTT dithiothreithol or TCEP
9. 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 ali quots 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 RA3 Add the indicated volume of 96 100 ethanol see table below to Buffer RA3 Concentrate Mark the label of the bottle to indicate that ethanol was added Store Wash Buffer RA3 at room temperature 18 25 C for up to one year MACHEREY NAGEL 01 2010 Rev 11 15 Total RNA Isolation NucleoSpin RNA II 10 preps 20 preps 50 preps 250 preps Cat No 740955 10 740955 20 740955 50 740955 250 Wash 5 ml 5 ml 12 5 ml 3 x 25 ml Buffer RA3 Add 20 ml Add 20 ml Add 50 ml Add 100 ml Concentrate ethanol ethanol ethanol ethanol to each vial rDNase 1 vial size C 1 vial size C 1 vial size D 5 vials size D RNase free Add 230 ul Add 230 ul Add 540 ul Add 540 ul lyophilized RNase free RNase free RNase free RNase free H O H O H O H O to each vial NucleoSpin RNA L 20 preps Cat No 740962 20 Wash Buffer RA3 25ml Concentrate rDNase RNase free lyophilized Add 100 ml ethanol 1 vial size D Add 540 ul RNase free H O 16 MACHEREY NAGEL 01 201
10. pure RNA with an Asd Azs ratio generally exceeding 1 9 measured in TE buffer pH 7 5 Even biological samples which are sometimes difficult to process will give high quality RNA Such samples are for example mouse tissue liver brain different tumor cell lines Streptococci and Actinobacillus pleuropneumoniae The isolated RNA is ready to use for applications like reverse transcriptase PCR RT PCR primer extension or RNase protection assays RNA of high integrity can be isolated with NucleoSpin RNA kits RIN RNA Integrity Number of RNA isolated from fresh high quality sample material e g eukaryotic cells or fresh mouse liver generally exceeds 9 0 However RNA integrity strongly depends on the sample quality RNA integrity was examined using the Agilent 2100 Bioanalyzer in conjunction with the RNA 6000 Nano or Pico assay The amount of DNA contamination is significantly reduced during on column digestion with rDNase Anyhow in very sensitive applications it might be possi ble to detect traces of DNA The NucleoSpin RNA II on column DNA removal is tested with the following procedure One million HeLa cells are subjected to RNA isolation according to the protocol RNA eluate is used as template for PCR detection of a 1 kb fragment in a 30 cycle reaction Generally no PCR fragment is obtained if the DNase is applied while a strong PCR fragment may be obtained if the DNase digestion is omitted The probability of DNA detection
11. 0 Rev 11 Total RNA Isolation 4 Safety instructions risk and safety phrases The following components of the NucleoSpin RNA Il and NucleoSpin RNA L kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section Component Hazard Hazard Risk Safety contents symbol phrases phrases rDNase rDNase x Xi May cause sensitiza R 42 43 S 22 24 RNase free lyophillized tion by inhalation and skin contact RA1 Guanidine x Xn Harmful by inhalation R S 19 thiocyanate in contact with skin 20 21 22 and if swallowed RA2 Guanidine x Xn Flammable Harmful R 10 S 13 16 thiocyanate by inhalation in 20 21 22 contact with skin and if swallowed MDB Guanidine Flammable R10 S 16 thiocyanate lt 10 etha nol lt 10 Risk phrases R10 Flammable R 20 21 22 Harmful by inhalation in contact with skin and if swallowed R 42 43 May cause sensitisation by inhalation and skin contact Safety phrases 13 Keep away from food drink and animal feedstuffs S 16 Keep away from sources of ignition No Smoking S 22 Do not breathe dust S24 Avoid contact with the skin Hazard labeling not neccessary if quantity per bottle below 25g or ml certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 8 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet Hazard labeling not neccessary if qu
12. 48 10 50 250 10 50 250 NucleoSpin RNA XS 740902 10 50 250 10 50 250 NucleoSpin RNA Clean up XS 740903 10 50 250 10 50 250 NucleoSpin RNA Buffer Set Buffer RA1 Buffer RA1 rDNase Set NucleoSpin Filters Collection Tubes 2 ml Visit www mn net com for more detailed product information 740944 740961 740961 500 740963 740606 740600 Suitable for 100 preps 50 ml 500 ml 1 set 50 1000 DISTRIBUTION AND USE OF NUCLEOSPIN RNA DNA BUFFER SET AND NUCLEOSPIN TRIPREP IN THE USA IS PROHIBITED FOR PATENT REASONS 42 MACHEREY NAGEL 01 2010 Rev 11 Total RNA Isolation 6 3 Product use restriction warranty NucleoSpin RNA kit components were developed designed distributed and sold FOR RESEARCH PURPOSES ONLY They are suitable FOR IN VITRO USES ONLY No claim or representation is intended for its use to identify any specific organism or for clinical use diagnostic prognostic therapeutic or blood banking It is rather the responsibility of the user to verify the use of the NucleoSpin RNA kit for a specific application range as the performance characteristic of this kit has not been verified to a specific organism This MACHEREY NAGEL product is shipped with documentation stating specifica tions and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL Ss sole obligation and the customer s sole remedy is limited to replaceme
13. Thawing of undisrupted animal tissue should be exclusively done in the presence of Buffer RA1 during simultaneous mechanical disruption e g with a rotor stator homogenizer This ensures that the RNA is not degraded by RNases before the prep aration has started The spinning rotor disrupts and simultaneously homogenizes the sample by mechanical shearing of DNA within seconds up to minutes homogenization MACHEREY NAGEL 01 2010 Rev 11 18 Total RNA Isolation time depends on sample Take care to keep the rotor tip submerged in order to avoid excess foaming Select a suitably sized homogenizer 5 7 mm diameter rotors can be used for homogenization in microcentrifuge tubes have to be incubated in lysozyme or lyticase zymolase solutions respectively see support protocols in section 5 By this treatment the robust cell walls of these organisms are digested or at least weakened which is essential for effective cell lysis by Buffer RA1 For microorganisms with extremely resistant cell walls like some Gram positive bacterial strains it may be necessary to optimize the conditions of the treatment with lytic enzymes or the cultivation conditions After lysis homogeni zation is achieved by the use of a NucleoSpin Filter or the syringe needle method 2 4 Elution procedures It is possible to adapt elution method and volume of water used for the subsequent application of interest In addition to the standard method described in th
14. allow full lysis activity of the lysis buffer To trypsinize adherent growing cells Aspirate cell culture medium and add an equal amount of PBS in order to wash the cells Aspirate PBS Add 0 1 0 3 trypsin in PBS and incubate for an appropriate time to detach the cells from the dish surface After cell detachment add medium transfer cells to an appropriate tube not supplied and pellet by centrifugation for 5 min at 300 x g Remove supernatant and continue with the addition of lysis buffer to the cell pellet are often solid and must therefore be broken up mechanically as well as lysed Depending on the disruption method the viscosity of the lysed sample has to be reduced further for optimal results It is essential for efficient RNA preparation that all the RNA contained in the sample is released from the cells by disruption and that the viscosity of the sample is reduced by homogenization The most commonly used technique for disruption of animal tissues is grinding with a pestle and mortar Grind the sample to a fine powder in the presence of liquid N Take care that the sample does not thaw during or after grinding or weighing and add the frozen powder to an appropriate aliquot of Buffer RA1 containing reducing agent e g B mercaptoethanol DTT or TCEP and mix immediately The broken up tissue must then be homogenized with a NucleoSpin Filter Filter L included in the kit or by passing 5 through a 0 9 mm syringe needle
15. ant It may be necessary to optimize incubation time and lyticase zymolase concentration depending on the yeast strain Continue with step 2 OR B Homogenization by mechanical disruption Harvest 2 5 ml of YPD culture 5 000 x g 10 min and wash with ice cold water Resuspend the cell pellet in a mixture of 350 ul Buffer RA1 and 3 5 ul f3 mercaptoethanol Add glass beads e g 300 mg glass beads 425 600 um Sigma Aldrich 68772 MACHEREY NAGEL 01 2010 Rev 11 25 NucleoSpin RNA II Shake samples in a swing mill at 30 Hz for 15 min Continue with step 3 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA 2 Lyse cells Add 350 ul Buffer RA1 and 3 5 pl B mercaptoethanol and vortex vigorously to lyse spheroplasts For appropriate sample and lysis buffer amounts see section 2 2 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA 3 Filtrate lysate Reduce viscosity and turbidity of the solution by filtration through NucleoSpin Filters violet rings Place NucleoSpin Filters in Collection Tubes 2 ml apply mixture and centrifuge for 1 min at 11 000 x g In case of visible pellet formation depending on sample amount and nature transfer supernatant without any formed pellet to a new 1 5 ml
16. antity per bottle below 125g or ml certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 8 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet MACHEREY NAGEL 01 2010 Rev 11 17 NucleoSpin RNA II 5 Protocols 5 1 Total RNA purification from cultured cells and tissue with NucleoSpin RNA II Before starting the preparation e Check if Wash Buffer RA3 and rDNase were prepared according to section 3 1 Homogenize sample Disrupt up to 30 mg of tissue for sample amounts see section 2 2 for homogenization methods see section 2 3 c Disrupt Up to 5 x 105 eukaryotic cultured cells are collected sample by centrifugation and lysed by addition of Buffer RA1 directly For appropriate sample and lysis buffer amounts see section 2 Lyse cells Add 350 pl Buffer RA1 and 3 5 pl B mercaptoethanol G ME to the cell pellet or to ground tissue and vortex vigorously 350 ul RA1 For appropriate sample and lysis buffer amounts see section 3 5 ul B ME 2 2 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 18 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin RNA II Filtrate lysate Reduce viscosity and clear the lysate by filtration through NucleoSpin Filter violet ring Place NucleoSpin Filter in a Collection Tube
17. ase zymolase concentration depending on the yeast strain Continue with step 2 OR B Homogenization by mechanical disruption Harvest an appropriate amount of cells from YPD culture 5 000 x g 10 min and wash with ice cold water Resuspend the cell pellet in a mixture of 3 6 ml Buffer RA1 and 36 ul B mercaptoethanol Add glass beads e g 300mg glass beads 425 600 um Sigma Aldrich 68772 32 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin RNA L Shake samples in a swing mill at 30 Hz for 15 min Continue with step 3 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 Lyse cells Add 3 6 ml Buffer RA1 and 36 pl B mercaptoethanol and vortex vigorously to lyse spheroplasts For appropriate sample and lysis buffer amounts see section 2 2 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 Filtrate lysate Reduce viscosity and turbidity of the solution by filtration through NucleoSpin Filter L Place NucleoSpin Filter L placed in Collection Tubes and centrifuge for 10 min at 4 500 x g In case of visible pellet formation depending on sample amount and nature transfer supernatant without any formed pellet to a new 15 ml centrifuge tube not supplied Adjust RNA binding conditions Discar
18. ated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written state ments signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty MACHEREY NAGEL 01 2010 Rev 11 43 Total RNA Isolation 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 Please contact MACHEREY NAGEL Germany Tel 49 0 24 21 969 270 e mail TECH BIO Q mn net com Last updated 12 2006 Rev 02 Trademarks RNA ater is a reg
19. be processed After addition of ethanol a stringy precipitate may become visible which will not affect the RNA isolation Be sure to mix thoroughly and apply sample as homogenious solution onto the column 34 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin RNA II NucleoSpin RNA L Proceed with step 5 8 and 9 of the NucleoSpin RNA II standard protocol section 5 1 or with step 5 8 and 9 of NucleoSpin RNA L standard protocol section 5 6 Steps 6 and 7 of the respective protocols may be omitted in this case As alternative products for RNA clean up NucleoSpin RNA Clean up and NucleoSpin RNA Clean up XS are recommended see ordeering information MACHEREY NAGEL 01 2010 Rev 11 35 NucleoSpin RNA II NucleoSpin RNA L 5 10 Support protocol NucleoSpin RNA II and NucleoSpin RNA L Total RNA preparation from RNA ater treated samples Before starting the preparation e Check if Wash Buffer RA3 and rDNase were prepared according to section 3 1 Prepare sample Remove RNAl ate solution Cut an appropriate amount of tissue 2 Lysecells Add 350 pl NucleoSpin RNA 11 1 8 ml NucleoSpin RNA L Buffer RA1 and 3 5 ul NucleoSpin RNA 11 18 pl NucleoSpin RNA L B mercaptoethanol to the sample Disrupt the sample material by using for example rotor stator homogenizers for homogenization methods see section 2 4 Proceed with step 3 filtrate lysate of the NucleoSpin RNA II s
20. by user e Reducing agent B mercaptoethanol or DTT dithiothreithol or TCEP BisTris Bis 2 hydroxyethyl imino tris hydroxymethyl methane e Sorbitol and lyticase or zymolase for homogenization by enzymatic digestion or a swing mill and glass beads for homogenization by mechanical disruption Before starting the preparation e Check if Wash Buffer RA3 and rDNase were prepared according to section 3 1 Homogenize sample Two alternative protocols are given for homogenization of yeast cells Users may choose between an enzymatic digestion A or mechanical homogenization B depending on laboratory equipment and personal preference Homogenization by enzymatic digest is only recommended for fresh harvested cells homogeni zation by mechanical disruption may also be performed with yeast cell pellets stored at 70 C for several months Note Due to the much higher concentration of genome equivalents in a nucleic acid preparation of yeasts compared with cultured cells or tissue material it may be nec essary to use a lower quantity of cells for the preparation A Homogenization by enzymatic digest Harvest 2 5 ml of YPD culture 5 000 x g 10 min Resuspend pellet in an appropriate amount of fresh prepared sorbitol lyticase buffer 50 100 U lyticase or zymolase in 1 M sorbitol 100 mM EDTA and incubate at 30 C for 30 min Pellet the resulting spheroplasts by centrifugation 1 000 x g 10 min Carefully discard suprnat
21. ction Tube Buffer RA2 will inactivate the rDNase Add 2 6 ml Buffer RA3 to the NucleoSpin RNAL Column Centrifuge for 3 min at 4 500 x g The flow through has not to be discarded in this step Leave the NucleoSpin RNA L Column in the Collection Tube Add 2 6 ml Buffer RA3 to the NucleoSpin RNAL Column Centrifuge for 5 min at 4 500 x g to dry the membrane completely Place the column into a fresh Collection Tube 15 ml supplied 9 Elute RNA Pipette 500 pl RNase free H O supplied directly onto the center of the silica membrane Incubate at room temperature for 2min and centrifuge for 3min at 4 500 x g Reduction of elution volume will generally not result in an increased concentration of eluted nucleic acid with the NucleoSpin RNA L kit see section 2 4 for alternative elution procedures q 6 250 ul rDNase reaction mixture RT 15 min 2 6 ml RA2 4 500 x g 3 min 2 6 ml RA3 4 500 x g 3 min 2 6 ml RA3 4 500 x g 5 min 500 ul RNase free H O RT 2 min 4 500 x g 3 min 30 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin RNA L 5 7 Support protocol NucleoSpin RNA L Total RNA preparation from up to 5 x 10 bacterial cells Additional reagent to be supplied by user e Lysozyme Before starting the preparation e Check if Wash Buffer RA3 and rDNase were prepared according to section 3 1 MHomogenize sample Resuspend the bacterial cell pellet Gram negative strains
22. d 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 Protein and DNA NucleoSpin RNA DNA Buffer Set NucleoSpin TriPrep The NucleoSpin RNA DNA Buffer Set see ordering information is a support set for RNA and DNA isolation in conjunction with NucleoSpin RNA II NucleoSpin RNA XS NucleoSpin RNA Plant or NucleoSpin RNA Protein 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 The combination of the NucleoSpin RNA DNA Buffer Set with NucleoSpin RNA Protein allows parallel isolation of RNA DNA and protein from one undivided sample The NucleoSpin TriPrep kit features the purification of RNA DNA and protein from single undivided samples DISTRIBUTION AND USE OF NUCLEOSPIN RNA DNA BUFFER SET AND NUCLEOSPIN TRIPREP IN THE USA IS PROHIBITED FOR PATENT REASONS 8 MACHEREY NAGEL 01 2010 Rev 11 Total RNA Isolation 2 2 Kit specifications NucleoSpin RNA kits are recommended for the isolation of total RNA from cultured cells and tissue Support protocols for the isolation of total RNA from cell free biological fluids bacteria and yeasts using the NucleoSpin RNA II kit are included The NucleoSpin RNA kits allow purification of
23. d the NucleoSpin Filter L and add 3 6 ml 70 ethanol to the lysate in the Collection Tube and mix by vortexing Proceed with step 5 of the NucleoSpin RNA L standard protocol section 5 6 MACHEREY NAGEL 01 2010 Rev 11 33 NucleoSpin RNA II NucleoSpin RNA L 5 9 Support protocol NucleoSpin RNA II and NucleoSpin RNA L Clean up of RNA from reaction mixtures Before starting the preparation Check if Wash Buffer RA3 was prepared according to section 3 Prepare sample Fill up RNA samples smaller than 100 pl with RNase free H O to 100 yl If different samples with varying volumes between 100 and 200 ul are purified RNA samples should be filled up with RNase free H O to a uniform volume e g 200 ul Prepare lysis binding buffer premix Prepare a Buffer RA1 ethanol premix with ratio 1 1 For each 100 ul RNA sample mix 300 ul Buffer RA1 and 300 yl ethanol 96 100 If multiple samples are processed the preparation of a master premix is recommended e g 2 ml RA1 2 ml 98 ethanol for approximately 6 prepara tions Filtrate lysate Not necessary Adjust RNA binding conditions To 100 pl of RNA sample add 600 pl 6 volumes of Buffer RA1 ethanol premix Mix sample with premix by vortexing If 200 ul of RNA samples are processed add 1200 ul of RA1 ethanol premix Maximal loading capacity of NucleoSpin RNA II Columns is 750 ul Repeat the procedure if larger volumes are to
24. e column in a new Collection Tube 2 ml Maximal loading capacity of NucleoSpin RNA II Columns is 750 ul Repeat the procedure if larger volumes are to be processed T 11 000 x g 1 min 350 ul 7096 ethanol Mix Load lysate 11 000 x g 30s MACHEREY NAGEL 01 2010 Rev 11 19 NucleoSpin RNA II Desalt silica membrane Add 350 pl MDB Membrane Desalting Buffer and centrifuge at 11 000 x g for 1 min to dry the membrane Salt removal will make the following rDNase digest much more effective If the column outlet has come into contact with the flow through for any reason discard the flow through and centrifuge again for 30 s at 11 000 x g Digest DNA Prepare DNase reaction mixture in a sterile 1 5 ml microcentrifuge tube not provided For each isolation add 10 pl reconstituted rDNase also see section 3 to 90 ul Reaction Buffer for rDNase Mix by flicking the tube Apply 95 pl DNase reaction mixture directly onto the center of the silica membrane of the column Incubate at room temperature for 15 min Wash and dry silica membrane Add 200 ul Buffer RA2 to the NucleoSpin RNA II Column Centrifuge for 30s at 11 000 x g Place the column into a new Collection Tube 2 ml Buffer RA2 will inactivate the rDNase Add 600 ul Buffer RA3 to the NucleoSpin RNA II Column Centrifuge for 30 s at 11 000 x g Discard flow through and place the column back into the Collection Tube No
25. e 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 of bound nucleic acid will be eluted High yield and high concentration Elute with the standard elution volume and apply the eluate once more onto the column for reelution Eluted RNA should immediately be put and always kept on ice for optimal stability because almost omnipresent RNases general lab ware fingerprints dust will degrade RNA For short term storage freeze at 20 C for long term storage freeze at 70 C 14 MACHEREY NAGEL 01 2010 Rev 11 Total RNA Isolation 3 Storage conditions and preparation of working solutions Attention Buffers RA1 RA2 and MDB contain guanidine thiocyanate Wear gloves and goggles Store lyophilized rDNase RNase free at 4 C on arrival stable up to 1 year All other kit components should be stored at room temperature 18 25 C and are stable up to one year Storage at lower temperatures may cause precipita tion of salts Check that 70 ethanol is available as additional solution to adjust RNA binding conditions in the lysate Check that reducing agent B ME DTT or TCEP is available Before starting any NucleoSpin RNA II protocol prepare the following rDNase RNase free Add indicated volume of RNase free H O see table below to the rDNase vial and
26. ed according to section 3 Homogenize sample Not necessary Lyse sample Add 350 ul Buffer RA1 and 3 5 ul B mercaptoethanol to 100 pl of sample and vortex vigorously For appropriate sample and lysis buffer amounts see section 2 2 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 Filtrate lysate Not necessary Adjust RNA binding conditions Add 350 ul of ethanol 70 to the lysate and mix by vortexing Proceed with step 5 of the NucleoSpin RNA II standard protocol section 5 1 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin RNA II 5 3 Support protocol NucleoSpin RNA II Total RNA preparation from up to 10 bacterial cells Additional reagent to be supplied by user Lysozyme Before starting the preparation Check if Wash Buffer RA3 and rDNase were prepared according to section 3 1 Homogenize sample Resuspend the bacterial cell pellet Gram negative strains in 100 pl TE buffer 10 mM Tris HCl 1 mM EDTA pH 8 containing 1 mg ml lysozyme by vigorous vortexing Incubate at 37 C for 10 min For preparation of RNA from Gram positive bacteria resuspend cells in 100 ul TE containing 2 mg ml lysozyme It may be necessary to optimize incubation time and lysozyme concentration depending on the bacterial strain Note Due to the much higher concentration of genome equivalent
27. ent Please see also Manchester K L 1995 Value of A A ratios for measure ment of purity of nucleic acids Biotechniques 19 208 209 Wilfinger W W Mackey K and Chomczyski P 1997 Effect of pH and ionic strength on the spectrophotometric assess ment of nucleic acid purity Biotechniques 22 474 481 MACHEREY NAGEL 01 2010 Rev 11 39 Total RNA Isolation Sample material Sample material not stored properly Whenever possible use fresh material If this is not possible flash freeze the samples in liquid N Samples should always be kept at 70 C Never PORE RNA allow tissues to thaw before addition of Buffer RA1 Perform quality or yield disruption of samples in liquid N continued ENS e Insufficient disruption and or homogenization of starting material Ensure thorough sample disruption and use NucleoSpin Filters Filters L for easy homogenization of disrupted starting material Carry over of guanidinium thiocyanate e Carefully load the lysate to the NucleoSpin RNA II Column Low A A and try to avoid a contamination of the upper part of the e 260 7 230 column and the column lid ratio e Make sure that residual Wash Buffer RA2 is washed away with Wash Buffer RAS This may be done by applying Buffer RAS to the inner rim of the column Sample material Clogged Too much starting material used Overloading may lead to NucleoSpin decreased overall yield Reduce amount of sample material C
28. eoSpin FFPE RNA kit Cat No 740969 see ordering information is recommended Please also refer to Annunziata Gloghini Barbara Canal Ulf Klein Luigino Dal Maso Tiziana Perin Riccardo Dalla Favera and Antonino Carbone RT PCR Analysis of RNA Extracted from Bouin Fixed and Paraffin Embedded Lymphoid Tissues J Mol Diagn 2004 6 290 296 as one example for customer modification of the support protocol mentioned above MACHEREY NAGEL 01 2010 Rev 11 27 NucleoSpin RNA L 5 6 Total RNA purification from cultured cells and tissue with NucleoSpin RNA L Before starting the preparation Check if Wash Buffer RA3 and rDNase were prepared according to section 3 Homogenize sample Disrupt up to 100 mg of tissue for homogenization methods see section 2 3 Up to 4x10 eukaryotic cultured cells are collected by centrifugation and lysed by addition of Buffer RA1 directly To choose an appropriate amount of starting material see section 2 2 Lyse cells Add 1 8 ml Buffer RA1 and 18 pl B mercaptoethanol G ME to the disrupted material in a 15 ml centrifuge tube not supplied and vortex vigorously For appropriate sample and lysis buffer amounts see section 22 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 Filtrate lysate Apply the lysate to a NucleoSpin Filter L placed in a Collection T
29. er of DNA copies per preparation single copy tar get plastidial mitochondrial target plasmid transfected into cells decreasing of PCR amplicon size Use larger PCR targets e g gt 500 bp or intron spanning primers if possible Use support protocol 5 11 for subsequent rDNase digestion in solution Suboptimal performance of RNA in downstream experiments Carry over of ethanol or salt Do not let the flow through touch the column outlet after the second Buffer RA3 wash Be sure to centrifuge at the corresponding speed for the respective time in order to remove ethanolic Buffer RA3 completely Checkif Buffer RA3 has been equilibratedto room temperature before use Washing at lower temperatures lowers efficiency of salt removal by Buffer RAS 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 MACHEREY NAGEL 01 2010 Rev 11 41 Total RNA Isolation 6 2 Ordering information Product Cat No Pack of NucleoSpin RNA II 740955 10 20 50 250 10 20 50 250 NucleoSpin RNA L 740962 20 20 NucleoSpin RNA Protein 740933 10 50 250 10 50 250 NucleoSpin TriPrep 740966 10 50 250 10 50 250 NucleoSpin FFPE RNA 740969 10 50 250 10 50 250 NucleoSpin RNA Clean up 7409
30. inant RNase free DNase rDNase in the NucleoSpin RNA kits facilitates such a digestion in solution in order to remove even traces of contaminat ing DNA A Digest DNA Reaction setup Add 6 ul Reaction Buffer for rDNase and 0 6 ul rDNase to 60 pl eluted RNA Alternatively premix 100 ul Reaction Buffer for rDNase and 10 pl rDNase and add 1 10 volume to one volume of RNA eluate Gently swirl the tube in order to mix the solution Spin down gently approx 1 s at 1 000 x g to collect every droplet of the solution at the bottom of the tube B Incubate sample Incubate for 10 min at 37 C MACHEREY NAGEL 01 2010 Rev 11 37 NucleoSpin RNA II NucleoSpin RNA L C Repurify RNA Repurify RNA with a suitable RNA cleanup procedure for example by use of the NucleoSpin RNA Clean up NucleoSpin RNA Clean up XS kits 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 thoroughly 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 70 ethanol Dry RNA pellet and resuspend RNA in RNase free H O 38 MACHEREY NAGEL
31. istered trademark of AMBION Inc NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH amp Co KG All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency or operation 44 MACHEREY NAGEL 01 2010 Rev 11
32. lds of total RNA isolation using NucleoSpin RNA L Sample Average yield 1 x 10 HeLa cells 20 ug 1 x 10 HeLa cells 160 ug 2 x 10 HeLa cells 330 ug 4 x 10 HeLa cells 620 ug 200 mg pig liver 450 ug 200 mg mouse liver 320 ug 12 MACHEREY NAGEL 01 2010 Rev 11 Total RNA Isolation 2 3 Handling preparation and storage of starting materials RNA is not protected against digestion until the sample material is flash frozen or disrupted in the presence of RNase inhibiting or denaturing agents Therefore it is important that samples are flash frozen in liquid N immediately and stored at 70 C or processed as soon as possible Samples can be stored in Lysis Buffer RA1 after disruption at 70 C for up to one year at 4 C for up to 24 hours or up to several hours at room temperature Frozen samples are stable up to 6 months Frozen samples in Buffer RA1 should be thawed slowly before starting with the isolation of total RNA Wear gloves at all times during the preparation Change gloves frequently Cultured animal cells are collected by centrifugation and directly lysed by adding Buffer RA1 according to step 2 of the standard protocol see sections 5 1 5 6 Cell lysis of adherent growing cells in a culture dish Completely aspirate cell culture medium and continue immediately with the addition of Lysis Buffer RA1 to the cell culture dish Avoid incomplete removal of the cell culture medium in order to
33. microcentrifuge tube not sup plied Alternatively the lysate may be passed gt 5 times through a 0 9 mm needle 20 gauge fitted to a syringe 4 Adjust RNA binding conditions Add 350 ul of ethanol 70 to the lysate and mix by vortexing Proceed with step 5 of the NucleoSpin RNA II standard protocol section 5 1 26 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin RNA II 5 5 Support protocol NucleoSpin RNA II Total RNA preparation from paraffin embedded tissue Additional reagent to be supplied by user e Xylene Before starting the preparation e Check if Wash Buffer RA3 and rDNase were prepared according to section 3 A Put 10 mg of finely minced tissue into a 1 5 ml microcentrifuge tube not pro vided Add 300 pl xylene and incubate 5 min with constant mixing at room tempera ture B Centrifuge at maximum speed 13 000 rpm for 3 min to pellet the tissue Discard the xylene C Repeat the steps A and B twice for a total of three xylene washes D Add 300 ul of 96 ethanol to the tube and incubate 5 min with constant mixing at room temperature E Centrifuge at maximum speed 13 000 rpm for 3 min to pellet the tissue Discard the ethanol F Repeat steps D and E for a total of two ethanol washes Continue with step 1 of the NucleoSpin RNA II standard protocol section 5 1 Note For high performance isolation of RNA from formalin fixed paraffin embedded tissue the Nucl
34. nt of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish an extra copy MACHEREY NAGEL does not warrant against damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or im proper or abnormal use of this product against defects in products or components not manufactured by MACHEREY NAGEL or against damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY 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 spe cial including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the st
35. olumn or use larger volume of Buffer RA1 Poor RNA e Insufficient disruption and or homogenization of starting quality material Ensure thorough sample disruption and use or yield NucleoSpin Filters Filters L for easy homogenization of dis rupted starting material Contamination of RNA with genomic DNA rDNase not active 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 Too much cell material used Reduce quantity of cells or tissue used 40 MACHEREY NAGEL 01 2010 Rev 11 Total RNA Isolation Contamination of RNA with genomic DNA continued DNA detection system too sensitive The amount of DNA contamination is effectively reduced during the on column digestion with rDNase Anyhow it can not be guaranteed that the purified RNA is 100 free of DNA therefore in very sensitive applications it might still be possible to detect DNA The NucleoSpin RNA II Plant system is checked by the following procedure One million HeLa cells are subjected to RNA isolation according to the protocol RNA eluate is used as 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 The probability of DNA detection with PCR increases with the numb
36. 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 refer encing while performing the purification procedure All technical literature is available on the internet at www mn net com MACHEREY NAGEL 01 2010 Rev 11 7 Total RNA Isolation 2 Product description 2 1 The basic principle One of the most important aspects in the isolation of RNA is to prevent degradation of the RNA during the isolation procedure With the NucleoSpin RNA methods cells are lysed by incubation in a solution containing large amounts of chaotropic ions This lysis buffer immediately inactivates RNases which are present in virtually all biologi cal materials and creates appropriate binding conditions which favor adsorption of RNA to the silica membrane Contaminating DNA which is also bound to the silica membrane is removed by an rDNase solution which is directly applied onto the silica membrane during the preparation RNase free rDNase is supplied with the kit 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 kits can be performed at room tempera ture The eluate however should be treated with care because RNA is very sensitive to trace contaminations of RNases often foun
37. s in a nucleic acid preparation of bacteria compared with eukaryotic material it may be necessary to use a lower quantity of cells for the preparation 2 Lysecells Add 350 pl Buffer RA1 and 3 5 pl B mercaptoethanol to the suspension and vortex vigorously For appropriate sample and lysis buffer amounts see section 2 2 Note As alternative to B ME the reducing agent DTT or TCEP may be used Use a final concentration of 10 20 mM DTT or TCEP within the Lysis Buffer RA1 3 Filtrate lysate Reduce viscosity and turbidity of the solution by filtration through NucleoSpin Filters violet rings Place NucleoSpin Filters in Collection Tubes 2 ml apply mixture and centrifuge for 1 min at 11 000 x g In case of visible pellet formation depending on sample amount and nature trans fer supernatant without any formed pellet to a new 1 5 ml microcentrifuge tube not supplied Alternatively the lysate may be passed gt 5 times through a 0 9 mm needle 20 gauge fitted to a syringe MACHEREY NAGEL 01 2010 Rev 11 23 NucleoSpin RNA II 4 Adjust RNA binding conditions Add 350 ul of ethanol 70 to the lysate and mix by vortexing Proceed with step 5 of the NucleoSpin RNA II standard protocol section 5 1 24 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin RNA II 5 4 Support protocol NucleoSpin RNA II Total RNA preparation from up to 5 x 10 yeast cells Additional reagents and components to be supplied
38. sufficient to perform all preparations with 600 ul If required additional Lysis Buffer RA1 can be ordered separately see ordering information MACHEREY NAGEL 01 2010 Rev 11 11 Total RNA Isolation NucleoSpin RNA L The kit can be used for preparing RNA from different amounts of sample material For optimal results the volume of Lysis Buffer RA1 protocol step 1 and of ethanol protocol step 3 should be adapted according to table 4 Table 4 Lysis adaptation Sample Amount Volume of Lysis Buffer RA1 Ethanol protocol step 1 protocol step 4 Cultured animal cells 5 x 106 2 x 10 1 8 ml 1 8 ml e g HeLa cells 2x10 5x107 3 6 ml 3 6 ml Ahimaltiss 30 100 mg 1 8 ml 1 8 ml 100 200 mg 3 6 ml 3 6 ml Bacteria 1x10 5 x 10 1 8 ml 1 8 ml 2 x 10 1 x 107 3 6 ml 3 6 ml Yeast Up to 3 x 10 3 6 ml 3 6 ml An additional loading step is required if 3 6 ml Buffer RA1 and ethanol is used If you isolate RNA from a certain kind of tissue the first time with the NucleoSpin RNA L kit we recommend starting with no more than 100 mg of tissue Depending on the nature of the tissue up to 200 mg can be processed Do not use more than 200 mg of tissue to avoid clogging of the column e Depending on sample type the average yield is around 70 400 ug total RNA see table 5 The A A ratio indicating purity of the RNA generally exceeds 1 9 280 Table 5 Overview on average yie
39. tandard protocol section 5 1 or NucleoSpin RNA L standard protocol section 5 6 36 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin RNA II NucleoSpin RNA L 5 11 Support protocol NucleoSpin RNA II and NucleoSpin RNA L rDNase digestion in solution The on column rDNase digestion in the standard protocol is already very efficient and thus resulting in minimal residual DNA This DNA will not be detectable in most down stream 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 Atypical example for such a demanding application is an RT PCR reaction in which the primer molecules do not differentiate between cDNA derived from RNA and contami nating genomic DNA Especially if high copy number targets are analyzed e g multi gene family mitochondrial plastidal or plasmid targets from transfections e the target gene is of a very low expression level e the amplicon is relatively small lt 200 bp DNA digestion in solution can efficiently destroy contaminating DNA However stringent RNase control and subsequent repurification of the RNA in order to remove buffer salts DNase and digested DNA are usually required The high quality recomb
40. te Make sure that residual buffer from the previous wash step is washed away with Buffer RA3 350 ul MDB 11 000 x g 1 min e 95 ul rDNase reaction mixture RT 15 min 200 pl RA2 11 000 x g 30s c5 600 yl RA3 11 000x g 30s 20 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin RNA II Add 250 ul Buffer RA3 to the NucleoSpin RNA II Column Centrifuge for 2 min at 11 000 x g to dry the membrane completely Place the column into a nuclease 250 pl RAS free Collection Tube 1 5 ml supplied o 11 000x g If for any reason the liquid level in the Collection Tube has 2 min reached the NucleoSpin RNA II Column after centrifugation discard flow through and centrifuge again Note Make sure that residual buffer from the previous wash step is washed away with Buffer RAS Elute RNA Elute the RNA in 60 pl RNase free H O supplied and 60 yl centrifuge at 11 000 x g for 1 min RNase free H O If higher RNA concentrations are desired elution can be done with 40 ul Overall yield however will decrease when j 11 000x g using smaller volumes 1 min For further alternative elution procedures see section 2 4 MACHEREY NAGEL 01 2010 Rev 11 21 NucleoSpin RNA II 5 2 Support protocol NucleoSpin RNA II Total RNA preparation from biological fluids e g serum culture medium Before starting the preparation e Check if Wash Buffer RA3 and rDNase were prepar
41. ube and centrifuge sample for 10 min at 4 500 x g This step will homogenize the sample by re moval of residual insoluble material and simultaneous reduction of lysate viscosity In case of visible pellet formation depending on sample amount and nature transfer supernatant without any formed pellet to a new 15 ml centrifuge tube not supplied If working with small amounts of cultured cells e g 1 x 107 HeLa cells step 3 may be substituted by vigorous mixing of the sample Disrupt sample 1 8 ml RA1 18 pl B ME V e 4 500 x g 10 min 28 MACHEREY NAGEL 01 2010 Rev 11 NucleoSpin RNA L Adjust RNA binding conditions Discard the NucleoSpin Filter L and add 1 8 ml ethanol 7096 to the lysate in the Collection Tube and mix by vortexing 2 x 5 s use 3 6 ml of 70 ethanol if working with large sample amounts see step 2 After addition of ethanol a stringy precipitate may become visible which will not affect the further procedure Resuspend precipitates thoroughly before loading onto the NucleoSpin RNA L Column Bind RNA Load the lysate ethanol mixture maximal 3 8 ml onto a NucleoSpin RNA L Column Centrifuge for 3 min at 4 500 x g If working with large sample amounts apply the rest of the lysate ethanol mixture max 3 8 ml onto the column and centrifuge again If the lysate has not passed through the column centrifuge again at 4 500 x g for 10 min In case of column o
42. use lower quantities of sample e g 1 x 10 cultured cells or 10 mg of tissue resulting in about 20 ug of RNA The kit can be used for preparing RNA from different amounts of sample material For optimal results the volume of Lysis Buffer RA1 protocol step 1 and of ethanol protocol step 3 should be adapted according to Table 2 10 MACHEREY NAGEL 01 2010 Rev 11 Total RNA Isolation Table 2 Lysis adaptation Sample Amount Volume of Lysis Buffer RA1 Ethanol protocol step 1 protocol step 4 Cultured animal or human cells Up to 5 x 10 350 ul 350 ul e g HeLa cells Human or Up to 20 mg 350 ul 350 ul animal tissue 20 mg 30 mg 600 ul 600 ul Tissue stored Up to 20 mg 350 ul 350 ul in RNAlater 20 mg 30 mg 600 ul 600 ul Samples known 7 to be hard to lyse Upt 5X10 600 yl 600 ul An additional loading step is required if 600 ul Buffer RA1 and ethanol is used load the sample onto the column in two successive centrifugation steps e Depending on sample type the average yield is around 5 ug 70 ug total RNA see Table 3 The A sdAs ratio generally exceeds 1 9 indicating purity of the RNA NucleoSpin RNA II Sample Average yield 8 x 10 HeLa cells 1 5 ug 4 x 10 HeLa cells 4 ug 1 x 106 HeLa cells 14 ug 2 x 10 HeLa cells 21 ug 2 5 x 106 HeLa cells 25 ug 5 x 106 HeLa cells 50 ug The volume of Lysis Buffer RA1 included in the kit is not
43. verloading incomplete flow through of the sample might be observed e g the membrane is still wet or some lysate has not passed through Remove the lysate which has not passed through the column and continue with the next protocol step Use less starting material and carefully remove insoluble material in step 3 next time Desalt silica membrane Add 2 2 ml MDB Membrane Desalting Buffer to the NucleoSpin RNA L Column Centrifuge for 3 min at 4 500 x g Discard flow through If the silica membrane is not completely dry after centrifuga tion centrifuge again at 4 500 x g for 10 min This step will create optimal reaction conditions for the rDNase c V 6 1 8 ml 70 ethanol Mix Load max 3 8 ml lysate 4 500 x g 3 min 2 2 ml MDB 4 500 x g 3 min MACHEREY NAGEL 01 2010 Rev 11 29 NucleoSpin RNA L 7 Digest DNA Prepare DNase reaction mixture in a sterile microcen trifuge tube mix 235 pl Reaction Buffer for rDNase and 25 ul reconstituted rDNase see section 3 per NucleoSpin RNA L Column Mix thoroughly but gently Digest with rDNase Apply 250 pl DNase reaction mixture directly onto the center of the silica membrane Incubate at room temperature for 15 min 8 Wash and dry silica membrane Add2 6 ml Buffer RA2tothe NucleoSpin RNAL Column Incubate at room temperature for 2 min Centrifuge for 3 min at 4 500 x g Discard flow through and place the column back into the Colle
44. with PCR increases with 1 the number of DNA copies per preparation single copy target lt plastidial mitochondrial target plasmid transfected into cells 2 decreasing of PCR amplicon size MACHEREY NAGEL 01 2010 Rev 11 9 Total RNA Isolation Table 1 Kit specifications at a glance Parameter NucleoSpin RNA II NucleoSpin RNA L Sample material Typical yield Elution volume Binding capacity Preparation time Format Up to 5 x 106 cells Up to 30 mg tissue Up to 70 ug 40 120 ul 200 ug 30 min 6 preps Mini spin column Up to 5 x 10 cells Up to 200 mg tissue Up to 400 ug 500 ul 700 ug 80 min 4 preps Midi spin column NucleoSpin RNA II The standard protocol section 5 1 allows the purification of up to 70 ug of total RNA per NucleoSpin RNA II Column from up to 5 x 108 cultured cells or 30 mg of tissue also see table 1 The isolated RNA can be used as template in a RT PCR reaction Generally 1 1096 of the eluate of total RNA prepared from 1 x 108 cells or 10 mg of tissue is sufficient as template for RT PCR If possible intron spanning primers should be used for RT PCR The RNA prepared from such high amounts is generally free of residual DNA although minute traces of DNA may remain in the preparation if large amounts of material rich in nucleic acids are used However if the isolated RNA will be used as template in a RT PCR reaction we recommend to
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