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  • MNMACHEREY-NAGEL www.mn-net.com

    MACHEREY-NAGEL

    DIN EN ISO 9001: 2000CERTIFIED

    MNMACHEREY-NAGEL MACHEREY-NAGEL MN

    Total RNAPurificationfrom Plant

    User manualNucleoSpin RNA PlantNucleoSpin Blood L

    NucleoSpin Plant L

    NucleoSpin Plant XL

    April 2006/Rev. 03

  • Protocol at a glance (Rev. 03)Total RNA Purification from Plant

    Mini

    NucleoSpin RNA Plant

    1 Homogenization ofsample 100 mg

    2 Cell Lysis 350 l RA13.5 l -mercaptoethanol

    or

    350 l RAP3.5 l -mercaptoethanol

    Mix

    3 Filtration of lysate1 min

    11,000 x g

    4 Adjust RNAbinding conditions 350 l 70 % ethanol

    5 Bind RNA

    30 sec11,000 x g

    6 Desalt silicamembrane

    350 l MDB

    1 min11,000 x g

    7 Digest DNA 95 l DNase reaction mixture

    RT15 min

    8 Wash and Drysilica membrane

    1st wash 200 l RA2

    2nd wash 600 l RA3

    3rd wash 250 l RA3

    1st and 2nd30 sec

    11,000 x g

    3rd2 min

    11,000 x g

    60 l H2O (RNase free)9 Elute highly pure

    RNA

    1 min11,000 x g

    MACHEREY-NAGEL GmbH & Co. KG Neumann-Neander Str. 6-8 D-52355 Dren GermanyTel.: +49 (0) 24 21 969 270 Fax: +49 (0) 24 21 969 279 e-mail: [email protected]

  • Total RNA Purification from Plant

    MACHEREY-NAGEL 04/2006/ Rev 03 3

    Table of contents

    1 Kit contents 4

    2 Product description 5

    2.1 The basic principle 5

    2.2 About this user manual 6

    2.3 Kit specifications 6

    2.4 Handling, preparation, and storage of starting materials 7

    2.5 Elution procedures 8

    2.6 Yields with different samples 9

    3 Storage conditions and preparation of working solutions 11

    4 Safety Instructions risk and safety phrases 12

    5 Protocols 13

    5.1 Total RNA isolation from plant tissue or filamentous fungi 13

    5.2 Support protocol NucleoSpin RNA Plant: rDNase digestion in solution 17

    6 Appendix 19

    6.1 Troubleshooting 19

    6.2 Ordering information 22

    6.3 References 22

    6.4 Product use restriction / warranty 22

  • Total RNA Purification from Plant

    4 MACHEREY-NAGEL 04/2006/ Rev 03

    1 Kit contents

    NucleoSpin RNA Plant

    20 preps 50 preps 250 preps

    Cat. No. 740949.20 740949.50 740949.250

    Buffer RA1 10 ml 25 ml 125 ml

    Buffer RAP 10 ml 25 ml 125 ml

    Buffer RA2 15 ml 15 ml 80 ml

    Buffer RA3(concentrate) 5 ml 12.5 ml 3 x 25 ml

    Buffer MDB(Membrane DesaltingBuffer)

    10 ml 25 ml 125 ml

    Reaction buffer forrDNase

    3 ml 7 ml 35 ml

    rDNase, RNase-free(lyophilized)

    1 vial(size C)

    1 vial(size D)

    5 vials(size D)

    H2O (RNase-free) 5 ml 15 ml 65 ml

    NucleoSpin Filters(violet)

    20 50 250

    NucleoSpin RNAPlant columns (lightblue, plus collectingtube)

    20 50 250

    NucleoSpin

    collecting tubes60 150 750

    1.5-mlmicrocentrifuge tubes

    20 50 250

    Protocol 1 1 1

    For preparation of working solutions and storage conditions see section 3.

  • Total RNA Purification from Plant

    MACHEREY-NAGEL 04/2006/ Rev 03 5

    2 Product description

    2.1 The basic principle

    One of the most important aspects in the isolation of RNA is to prevent degradationof the RNA during the isolation procedure. With the NucleoSpin RNA Plantmethod, the cells are first disrupted by grinding in the presence of liquid N2. Completedenaturation is then achieved by incubation in a solution containing large amounts ofchaotropic ions. This lysis buffer immediately inactivates RNases which are presentin virtually all biological materials and creates appropriate binding conditions whichfavor adsorption of RNA to the silica membrane. Contaminating DNA, which is alsobound to the silica membrane, is removed by an rDNase solution which is directlyapplied onto the silica membrane during the preparation (RNase-free rDNase issupplied with the kit). Simple washing steps with two different buffers remove salts,metabolites and macromolecular cellular components. Pure RNA is finally elutedunder low ionic strength conditions with RNase-free water (supplied).

    The NucleoSpin RNA Plant kit contains two different lysis buffers, RA1 (guanidinethiocyanate) and RAP (guanidine-HCl), respectively. In most cases, use of bufferRA1 is recommended for lysis due to the stronger denaturing properties of thethiocyanate. The presence of peculiar metabolites in a variety of plant tissues orfungi, however, requires the use of an alternative buffer, because they may lead tosolidification of the lysate, resulting in a non-processible slurry. In such cases, RAP isthe buffer of choice.

    The RNA preparation using NucleoSpin RNA Plant kit can be performed at roomtemperature. The eluate, however, should be treated with care because RNA is verysensitive to trace contaminations of RNases, often found on general lab ware,fingerprints and dust. To ensure RNA stability keep RNA frozen at 20C for short-term or 70C for long-term storage.

    Simultaneous Isolation of RNA, Protein, and DNA (NucleoSpin RNA/DNAbuffer set)

    The NucleoSpin RNA/DNA buffer set (see ordering information) is a support set forRNA and DNA isolation in conjunction with NucleoSpin RNA II, NucleoSpin RNAPlant, or NucleoSpin RNA/Protein.

    This patented technology enables successive elution of DNA and RNA from oneNucleoSpin column with low salt buffer and water respectively. DNA and RNA areimmediately 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 undividedsample.

  • Total RNA Purification from Plant

    6 MACHEREY-NAGEL 04/2006/ Rev 03

    2.2 About this user manual

    Experienced users who are performing the isolation of total RNA using aNucleoSpin RNA Plant isolation kit may refer to the Protocol-at-a-glance instead ofthis User Manual. The Protocol-at-a-glance is designed to be used only as asupplemental tool for quick referencing while performing the purification procedure.First-time users are strongly advised to read this User Manual.

    2.3 Kit specifications

    NucleoSpin RNA Plant is recommended for the isolation of total RNA fromplant cells and tissues or filamentous fungi.

    Generally, 1 10 % of the eluate of total RNA prepared from 10 mg of planttissue is sufficient as template for RT-PCR*. If possible, intron-spanningprimers should be used for RT-PCR*. Hands-on time for RNA preparation fromplant tissue with NucleoSpin RNA Plant is less than 30 min.

    NucleoSpin Filters for homogenization and reduction of of lysate viscosityare included in the kit.

    The kit allows purification of up to 70 g of pure RNA, suitable for applicationslike reverse transcriptase-PCR* (RT-PCR*), Northern blotting, primerextension, or RNase protection assays.

    The amount of DNA contamination is significantly reduced during on-columndigestion with rDNase. Anyhow, in very sensitive applications it might bepossible to detect traces of DNA. The NucleoSpin RNA II system is checkedby following procedure: One million HeLa cells are subjected to RNA isolationaccording to the protocol. RNA eluate is used as template for PCR detection ofa 1kb fragment in a 30 cycle reaction. Generally, no PCR fragment is obtainedif the DNase is applied. However, a strong PCR fragment is obtained if DNaseis omitted. The eventuality of DNA detection with PCR increases with: 1. thenumber of DNA copies per preparation: single copy target < plastidial/mitochondrial target < plasmid transfected into cells, 2. decreasing of PCRamplicon size.

    * PCR is patented by Roche Diagnostics

  • Total RNA Purification from Plant

    MACHEREY-NAGEL 04/2006/ Rev 03 7

    Table 1: Kit specifications at a glance

    RNA Plant

    Sample size up to 100 mg tissue

    Average yield up to 70 g

    Elution volume 60 l

    Binding capacity 100 g

    Time/prep 30 min/ 6 preps

    Spin column type mini

    2.4 Handling, preparation, and storage of startingmaterials

    RNA is not protected against digestion until the sample material is flash frozen ordisrupted in the presence of RNase inhibiting or denaturing agents. Therefore it isimportant that samples are flash frozen in liquid N2 immediately and stored at 70Cor processed as soon as possible. Samples can be stored in lysis buffer RA1 afterdisruption at 70C for up to one year, at +4C for up to 24 hours or up to severalhours at room temperature. Frozen samples are stable up to 6 months. Frozensamples in buffer RA1 should be thawed slowly before starting with the isolation oftotal RNA.

    Wear gloves at all times during the preparation. Change gloves frequently.

    Plant tissues are often solid and must therefore be broken up mechanically as wellas lysed. Depending on the disruption method, the viscosity of the lysed sample hasto be reduced further for optimal results. It is essential for efficient RNA preparationthat all the RNA contained in the sample is released from the cells by disruption andthat the viscosity of the sample is reduced by homogenization.

    The most commonly used technique for disruption of plant tissues is grinding with apestle and mortar. Grind the sample to a fine powder in the presence of liquid N2.Take care that the sample does not thaw during or after grinding or weighing and addthe frozen powder to an appropriate aliquot of buffer RA1 respectively RAPcontaining -mercaptoethanol and mix immediately. The broken-up tissue must thenbe homogenized with a NucleoSpin Filter unit or by passing 5 through a 0.9 mmsyringe needle.

    Thawing of undisrupted plant tissue should be exclusively done in the presence ofbuffer RA1 during simultaneous mechanical disruption, e.g. with a rotor-stator

  • Total RNA Purification from Plant

    8 MACHEREY-NAGEL 04/2006/ Rev 03

    homogenizer. This ensures that the RNA is not degraded by RNases before thepreparation has started. The spinning rotor disrupts and simultaneously homogenizesthe sample by mechanical shearing of DNA within seconds up to minutes(homogenization time depends on sample). Take care to keep the rotor tipsubmerged in order to avoid excess foaming. Select a suitably sized homogenizer (5-7 mm diameter rotors can be used for homogenization in microcentrifuge tubes).

    2.5 Elution procedures

    It is possible to adapt elution method and volume of water used for the subsequentapplication of interest. In addition to the standard method described in the individualprotocols (recovery rate about 70-90%) there are several modifications possible.

    High yield: Perform two elution steps with the volume indicated in theindividual protocol. About 90-100% of bound nucleic acid will be eluted.

    High yield and high concentration: Elute with the standard elution volumeand apply the eluate once more onto the column for reelution.

    Eluted RNA should immediately be put and always kept on ice for optimal stabilitybecause almost omnipresent RNases (general lab ware, fingerprints, dust) willdegrade RNA. For short-term storage freeze at 20C, for long-term storage freezeat 70C.

  • Total RNA Purification from Plant

    MACHEREY-NAGEL 04/2006/ Rev 03 9

    2.6 Yields with different samples

    Typical yields of total RNA per 50 mg sample

    Species Organ Yield [g]

    Cucumis melo (cucumber) leaf 15

    Taraxacum officinale(dadelion)

    leaf 10

    Mucor rouxii (fungus) mycelium 6

    Allium sativum (garlic) leaf 13

    Zea mays (maize) leaf 18

    Allium cepa (onion) germ bud 13

    Brassica napus (rapeseed)

    leaf

    blossom

    stalk

    9

    9

    7

    Capsicum annuum (redpepper)

    leaf 8

    Secale cereale (rye) leaf 12

    Beta vulgaris (sugar beet) leaf 17

    Thymus herba-barona(thyme)

    leaf 15

    Nicotiana tabacum (tabacco)

    Leaf

    root tip

    stalk

    blossom

    24

    12

    18

    33

    Lycopersicum esculentum(tomato)

    leaf 10

    Viola tricolor (viola) leaf 9

    Triticum aestivum (wheat) leaf 4

  • Total RNA Purification from Plant

    10 MACHEREY-NAGEL 04/2006/ Rev 03

    Species Organ Yield [g]

    Lactuca sativa (lettuce) leaf 4

    Gladiolus spec. leaf 7

    Hordeum vulgare (barley) leaf 3

    Arabidopsis thaliana(Thale cress)

    leaf 15

  • Total RNA Purification from Plant

    MACHEREY-NAGEL 04/2006/ Rev 03 11

    3 Storage conditions and preparation of workingsolutions

    Attention:

    Buffers RA1, RA2 and MDB contain guanidine thiocyanate; RAP contains guanidine-HCl. Wear gloves and goggles!

    Store lyophilized RNase-free rDNase at +4C on arrival (stable up to oneyear).

    All other kit components should be stored at room temperature (20-25C) andare stable up to one year. Storage at lower temperatures may causeprecipitation of salts.

    Check that 70% ethanol is available as additional solution in the lab to adjustRNA binding conditions in the buffer RA1 or RAP lysate.

    Before you start any NucleoSpin RNA Plant protocol prepare the following:

    RNase-free rDNase: Add indicated volume of RNase-free water to therDNase vial and incubate for 1 min at room temperature. Gently swirl the vialsto completely dissolve the rDNase. Be careful not to mix rDNase vigorously.rDNase is sensitive to mechanical agitation. Dispense into aliquots and storeat 18C. The frozen working solution is stable for 6 months. Do notfreeze/thaw the aliquots more than three times.

    Buffer RA3: Add the indicated volume of ethanol (96 100%) to the bufferRA3 concentrate. Store buffer RA3 at room temperature (20-25C) for up toone year.

    NucleoSpin RNA Plant

    20 preps 50 preps 250 preps

    Cat. No. 740949.20 740949.50 740949.250

    Buffer RA3(concentrate)

    5 mladd 20 ml ethanol

    12.5 mladd 50 ml ethanol

    3 25 mladd to each bottle

    100 ml ethanol

    rDNase, RNase-free(lyophilized)

    1 vial(size C)

    add 230 lRNase-free water

    1 vial(size D)

    add 540 lRNase-free water

    5 vials(size D)

    add 540 lRNase-free water

    to each vial

  • Total RNA Purification from Plant

    12 MACHEREY-NAGEL 04/2006/ Rev 03

    4 Safety Instructions risk and safety phrases

    The following components of the NucleoSpin RNA Plant kits contain hazardouscontents.

    Wear gloves and goggles and follow the safety instructions given in this section.

    Component HazardContents

    HazardSymbol

    RiskPhrases

    SafetyPhrases

    rDNase rDNase,lyophilized

    Xn May cause sensitization by inhalationand skin contact

    R 42/43 S 22-24

    Buffer RA1 guanidinethiocyanate

    Xn Harmful by inhalation, in contact with

    skin and if swallowedR 20/21/22 S 13

    Buffer RA2 guanidinethiocyanate

    Xn Harmful by inhalation, in contact with

    skin and if swallowedR 20/21/22 S 13

    Buffer RAP guanidinehydrochloride

    Xn Harmful if swallowed.

    Irritating to eyes and skinR 22-36/38 S 22

    MDB guanidinethiocyanate< 10%

    Substance does not have to be specially labeled as hazardous

    Risk Phrases

    R 22 Harmful if swallowed

    R 20/21/22 Harmful by inhalation, in contact with the skin and if swallowed

    R 36/38 Irritating to eyes and skin

    R 42/43 May cause sensitisation by inhalation and skin contact

    Safety Phrases

    S 13 Keep away from food, drink and animal feedstuffs

    S 22 Do not breathe dust

    S 24 Avoid contact with the skin

    Label not necessary, if quantity below 125 g or ml (concerning 67/548/EEC Art. 25, 1999/45/EC Art.

    12 and German GefStoffV 42 and TRGS 200 7.1)

  • NucleoSpin RNA Plant

    MACHEREY-NAGEL 04/2006/ Rev 03 13

    5 Protocols5.1 Total RNA isolation from plant tissue or filamentous

    fungi

    1 Homogenization of sample

    Grind up to 100 mg of tissue under liquid N2. (forhandling and preparation methods see section 2.4)

    Grind sample

    2 Cell lysis

    Add 350 l RA1 and 3.5 l -mercaptoethanol to100 mg ground tissue and vortex vigorously.

    If the lysate solidifies upon addition of RA1, use 350 lbuffer RAP instead.

    + 350 l RA1+ 3.5 l -ME

    or

    + 350 l RAP+ 3.5 l -ME

    3 Filtration of the lysate

    Reduce viscosity and clear the lysate by filtrationthrough NucleoSpin Filter units: Place NucleoSpin

    Filter (violet) units in collecting tubes, apply the mixture,and centrifuge for 1 min at 11,000 x g. Transfer thefiltrate to a new microcentrifuge tube.

    Important note:

    Do not disturb the pellet of cell debris at the bottom of thecollecting tube, which may be visible after centrifugation.

    In case of visible pellet formation (depending on sampleamount and nature) transfer supernatant without anyformed pellet to a new 1.5 ml centrifuge tube (notincluded).

    1 min11,000 x g

  • NucleoSpin RNA Plant

    14 MACHEREY-NAGEL 04/2006/ Rev 03

    4 Adjust RNA binding conditions

    Discard the NucleoSpin Filter unit and add 350 lethanol (70%) to the recovered filtrate resulting fromstep 3. Mix by pipetting up and down (5 times).

    Alternatively transfer flow-through into a new 1.5 ml tube(not provided), add 350 l ethanol (70%), and mix byvortexing (2 x 5 sec).

    After addition of ethanol a stringy precipitate may becomevisible which will not affect the RNA isolation. Be sure todisaggregate any precipitate by mixing and load all of theprecipitate on the column as described in step 5. Do notcentrifuge the ethanolic lysate before loading it onto thecolumn in order to avoid pelleting the precipitate.

    + 350 l 70%ethanol

    mix

    5 Bind RNA

    For each preparation, take one NucleoSpin RNA Plantcolumn (light blue) placed in a 2 ml centrifuge tube andload the lysate. Centrifuge for 30 s at 11,000 x g.Place the column in a new collecting tube.

    Maximal loading capacity of NucleoSpin RNA Plant columnsis 750 l. Repeat the procedure if larger volumes are to beprocessed.

    load lysate

    30 s11,000 g

    6 Desalt silica membrane

    Add 350 l MDB (Membrane Desalting Buffer) andcentrifuge at 11,000 x g for 1 min to dry the membrane.

    Salt removal will make the following digest with rDNase muchmore effective. If the column outlet has come into contact withthe flow-through for any reason, discard the flow-through andcentrifuge again for 30 s at full speed (max. 11,000 x g).

    + 350 l MDB

    1 min11,000 x g

  • NucleoSpin RNA Plant

    MACHEREY-NAGEL 04/2006/ Rev 03 15

    7 Digest DNA

    Prepare DNase reaction mixture in a sterilemicrocentrifuge tube: for each isolation, add 10 lreconstituted rDNase (see section 3) to 90 l Reactionbuffer for rDNase. Mix by flicking the tube.

    Apply 95 l DNase reaction mixture directly onto thecenter of the silica membrane of the column. Incubate atroom temperature for 15 min.

    + 95 l DNasesolution

    RT15 min

    8 Wash and Dry silica membrane

    1st wash

    Add 200 l RA2 to the NucleoSpin RNA Plant columnand centrifuge for 30 s at 11,000 g. Place the columninto a new collecting tube.

    Buffer RA2 will inactivate the rDNase.

    + 200 l RA2

    30 s11,000 g

    2nd wash

    Add 600 l RA3 to the NucleoSpin RNA Plant column.Centrifuge for 30 s at 11,000 g. Discard flow-throughand place the column back into the collecting tube.

    + 600 l RA3

    30 s11,000 g

    3rd wash

    Add 250 l RA3 to the NucleoSpin RNA Plant column.Centrifuge for 2 min at 11,000 x g to dry the membranecompletely. Place the column into a nuclease-free 1.5 mlmicrocentrifuge tube (supplied).

    If for any reason, the liquid level in the collecting tube hasreached the NucleoSpin RNA Plant column aftercentrifugation, discard the flow-through and centrifuge again.

    + 250 l RA3

    2 min11,000 x g

  • NucleoSpin RNA Plant

    16 MACHEREY-NAGEL 04/2006/ Rev 03

    9 Elute highly pure RNA

    Elute the RNA in 60 l RNase-free water (supplied) andcentrifuge at 11,000 x g for 1 min.

    If higher RNA concentrations are desired, elution can be donewith volumes as low as 40 l. Overall yield, however, willdecrease when using smaller elution volumes.

    For further alternative elution procedures see section 2.5.

    + 60 l H2O

    1 min11,000 x g

  • NucleoSpin RNA Plant

    MACHEREY-NAGEL 04/2006/ Rev 03 17

    5.2 Support protocol NucleoSpin RNA Plant: rDNasedigestion in solution

    The on-column rDNase digestion in the standard protocol is already very efficient andthus resulting in a minimal residual DNA content of the purified RNA. This DNA willnot be detectable in most downstream applications. Despite this, there are still certainapplications which require even lower contents of residual DNA. However, removal ofDNA to a completely undetectable level is challenging and the efficiency of an on-column DNA digestion is sometimes not sufficient for downstream applicationsrequiring lowest residual content of DNA.

    A typical example for such a demanding application is an RT-PCR reaction in whichthe primer molecules do not differentiate between cDNA (derived from RNA) andcontaminating 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 (

  • NucleoSpin RNA Plant

    18 MACHEREY-NAGEL 04/2006/ Rev 03

    3 Repurification of RNA

    Repurify RNA with a suitable RNA cleanup procedure, e.g. by use of theNucleoSpin RNA clean-up kit (see ordering information) or by ethanolprecipitation.

    Ethanol precipitation, exemplary

    Add 0.1 volume of 3M 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 20C or +4C.

    Note: chose 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 max. speed.

    Wash RNA pellet with 70% ethanol.

    Dry RNA pellet and resuspend RNA in RNase-free water.

  • Total RNA Purification from Plant

    MACHEREY-NAGEL 04/2006/ Rev 03 19

    6 Appendix6.1 Troubleshooting

    Problem Possible cause and suggestions

    RNA isdegraded/no RNAobtained

    RNase contamination

    Create an RNase free working environment. Wear gloves duringall steps of the procedure. Change gloves frequently. Use ofsterile, disposable polypropylene tubes is recommended. Keeptubes closed whenever possible during the preparation.Glassware should be oven-baked overnight at 250C before use.

    Poor RNAqualityor yield

    Reagents not applied or restored properly

    Reagents not properly restored. Add the indicated volume ofnuclease-free water to rDNase vial and 96% ethanol to bufferconcentrate RA3 and mix. Reconstitute and store lyophilizedrDNase according to instructions in section 3.

    Sample and reagents have not been mixed completely. Alwaysvortex vigorously after each reagent has been added.

    No ethanol has been added after lysis. Binding of RNA to thesilica membrane is only effective in the presence of ethanol.

    Kit storage

    Reconstitute and store lyophilized rDNase according toinstructions in section 3.

    Store other kit components at room temperature. Storage at lowtemperatures may cause salt precipitation.

    Keep bottles tightly closed in order to prevent evaporation orcontamination.

    Ionic strength and pH influence A260 absorption as well as ratio A260/280

    For adsorption measurement, use 5 mM Tris pH 8.5 as diluent.Please see also:- Manchester, K L. 1995. Value of A260/A280 ratios formeasurement of purity of nucleic acids. Biotechniques 19, 208-209.- Wilfinger, W W, Mackey, K and Chomczyski, P. 1997. Effect ofpH and ionic strength on the spectrophotometric assessment ofnucleic acid purity. Biotechniques 22, 474-481.

  • Total RNA Purification from Plant

    20 MACHEREY-NAGEL 04/2006/ Rev 03

    Problem Possible cause and suggestions

    Poor RNAqualityor yield(continued)

    Sample material

    Sample material not stored properly. Whenever possible, usefresh material. If this is not possible, flash freeze the samples inliquid N2. Samples should always be kept at 70C. Never allowtissues to thaw before addition of buffer RA1. Perform disruptionof samples in liquid N2.

    Insufficient homogenization of starting material. Always grind thesamples in the presence of liquid N2. Use NucleoSpin

    Filterunits.

    CloggedNucleoSpin

    column

    Sample material

    Too much starting material used. Overloading may lead todecreased overall yield. Reduce amount of sample or use largerRA1 volume.

    Insufficient disruption and/or homogenization of starting material.Ensure thorough sample disruption and use NucleoSpin Filterunits for easy homogenization of disrupted starting material.

    Contaminationof RNA withgenomic DNA

    rDNase not active

    Reconstitute and store lyophilized rDNase according toinstructions in section 3.

    DNase solution not properly applied

    Pipet rDNase solution directly onto the center of the silicamembrane.

    Too much cell material used

    Reduce quantity of cells or plant tissue used.

  • Total RNA Purification from Plant

    MACHEREY-NAGEL 04/2006/ Rev 03 21

    Problem Possible cause and suggestions

    Contaminationof RNA withgenomic DNA(continued)

    DNA detection system too sensitive

    The amount of DNA contamination is significantly reduced during theon-column digestion with rDNase. Anyhow it can not be guaranteedthat the purified RNA is 100% free of DNA, therefore in very sensitiveapplications it might be possible to detect DNA.The NucleoSpin RNA II/Plant system is checked by the followingprocedure: One million HeLa cells are subjected to RNA isolationaccording to the protocol. RNA eluate is used as template for PCRdetection of a 1kb fragment in a 30 cycle reaction.Generally, no PCR fragment is obtained if the DNase is applied,however, a strong PCR fragment is obtained if DNase is omitted.The eventuality of DNA detection with PCR increases with:

    - the number of DNA copies per preparation: single copy target 500bp) or intron spanning primers ifpossible.

    Use support protocol 5.5 for subsequent rDNase digestion insolution.

    Suboptimalperformanceof RNA indownstreamexperiments

    Carryover of ethanol or salt

    Do not let the flow-through touch the column outlet after thesecond RA3 wash. Be sure to centrifuge 1 min at full speed(max. 11,000 x g) in order to remove ethanolic buffer RA3completely.

    Check if buffer RA3 has been equilibrated to room temperaturebefore use. Washing at lower temperatures lowers efficiency ofsalt removal by RA3.

    Store isolated RNA properly

    Eluted RNA should always be kept on ice for optimal stabilitysince trace contaminations of omnipresent RNases (general labware, fingerprints, dust) will degrade the isolated RNA. For shortterm storage freeze at 20C, for long term storage freeze at70C.

  • Total RNA Purification from Plant

    22 MACHEREY-NAGEL 04/2006/ Rev 03

    6.2 Ordering information

    Product Cat. No. Pack of

    NucleoSpin RNA Plant 740949.20/.50/.250 20/50/250

    NucleoSpin RNA Clean-up 740948.10/.50/.250 10/50/250

    NucleoSpin RNA/Protein 740933.10/.50/.250 10/50/250

    NucleoSpin RNA/DNA buffer set 740944 suitable for 100 preps

    Lysis buffer RA1 740961 50 ml

    Lysis buffer RA1 740961.500 500 ml

    Wash buffer RA4 concentrate(for 1l)

    740960 300 ml

    rDNase set 740963 1 set

    NucleoSpin Filters 740606 50

    NucleoSpin collection tubes 740600 1000

    6.3 References

    Vogelstein B., and D. Gillespie. 1979. Preparative and analytical purification of DNAfrom agarose. Proc. Natl. Acad. Sci. USA 76: 615-619.

    6.4 Product use restriction / warranty

    NucleoSpin RNA Plant kits components were developed, designed and sold forresearch purposes only. They are suitable for in vitro uses only. No claim orrepresentation is intended for its use to identify any specific organism or for clinicaluse (diagnostic, prognostic, therapeutic, or blood banking).

    It is rather the responsibility of the user to verify the use of the NucleoSpin RNAPlant kits for a specific application range as the performance characteristic of this kithas not been verified to a specific organism.

    This MACHEREY-NAGEL product is shipped with documentation statingspecifications and other technical information. MACHEREY-NAGEL warrants to meetthe stated specifications. MACHEREY-NAGELs sole obligation and the customerssole remedy is limited to replacement of products free of charge in the event productsfail to perform as warranted. Supplementary reference is made to the general

  • Total RNA Purification from Plant

    MACHEREY-NAGEL 04/2006/ Rev 03 23

    business terms and conditions of MACHEREY-NAGEL, which are printed on theprice list. Please contact us if you wish an extra copy.

    MACHEREY-NAGEL does not warrant against damages or defects arising inshipping and handling (transport insurance for customers excluded), or out ofaccident or improper or abnormal use of this product; against defects in products orcomponents not manufactured by MACHEREY-NAGEL, or against damagesresulting from such non-MACHEREY-NAGEL components or products.

    MACHEREY-NAGEL makes no other warranty of any kind whatsoever, andSPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANYKIND OR NATURE WHATSOEVER, DIRECTLY OR INDIRECTLY, EXPRESS ORIMPLIED, INCLUDING, WITHOUT LIMITATION, AS TO THE SUITABILITY,REPRODUCTIVITY, DURABILITY, FITNESS FOR A PARTICULAR PURPOSE ORUSE, MERCHANTABILITY, CONDITION, OR ANY OTHER MATTER WITHRESPECT 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, orspecial (including but not limited to loss of use, revenue or profit), whether basedupon warranty, contract, tort (including negligence) or strict liability arising inconnection with the sale or the failure of MACHEREY-NAGEL products to perform inaccordance with the stated specifications. This warranty is exclusive andMACHEREY-NAGEL makes no other warranty expressed or implied.

    The warranty provided herein and the data, specifications and descriptions of thisMACHEREY-NAGEL product appearing in MACHEREY-NAGEL publishedcatalogues and product literature are MACHEREY-NAGELs sole representationsconcerning the product and warranty. No other statements or representations, writtenor oral, by MACHEREY-NAGELs employees, agent or representatives, exceptwritten statements signed by a duly authorized officer of MACHEREY-NAGEL areauthorized; they should not be relied upon by the customer and are not a part of thecontract of sale or of this warranty.

    Product claims are subject to change. Therefore please contact our TechnicalService 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 forinformational purposes only. MACHEREY-NAGEL does not warrant that allapplications have been tested in MACHEREY-NAGEL laboratories usingMACHEREY-NAGEL products. MACHEREY-NAGEL does not warrant thecorrectness of any of those applications.

    Please contact:

    MACHEREY-NAGEL GermanyTel.: +49 24 21 969-270e-mail: [email protected]