NucleoSpin® RNA XS - MACHEREY
Contents
1. RNA isolation User manual NucleoSpin RNA XS September 2015 Rev 09 MACHEREY NAGEL www mn net com SUN RNA isolation Protocol at a glance Rev 09 XS NucleoSpin RNA XS Use up to 10 cultured cells 1 Supply sample or 5 mg tissue samples TT 100 pL RA1 2 Lyse and 2 uL TCEP homogenize cells y Mix 5 uL Carrier RNA 3 Add Carrier RNA working solution Mix 4 Filtrate lysate E 11 000 x g optional y 30s I 5 Adjust RNA 100 uL 70 ethanol binding condition Mix S Load lysate 6 Bind RNA u 11 000 x g S7 30s 100 pL MDB 7 Desalt silica c membrane 11 000 x g g 30 s S 25 uL DNase 8 Digest DNA E reaction mixture RT 15 min 1 wash 100 uL RA2 RT 2 min 11 000 x g 30 s 9 Wash and dry 3rlayash 400 uL RAS silica membrane 11 000 x g 30 s 3 wash 200 uL RA3 11 000 x g 2 min 10 uL RNase free H O 10 Elute highly pure RNA 11 000 x g 30s MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 52355 D ren Germany Tel 49 24 21 969 270 Fax 49 24 21 969 199 tech bio mn net com www mn net com RNA isolation Table of contents 1 Components 4 1 1 Kit contents 4 1 2 Reagents consumables and equipment to be supplied by user 5 1 3 About this user manual 5 2 Product description 6 2 1 The basic principle 6 2 2 Kit specifications 6 2 3 Handling preparation and storage of starting2. 10 50 250 NucleoSpin RNA DNA Buffer Set 740944 Suitable for 100 preps Buffer RA1 740961 60 mL 740961 500 500 mL rDNase Set 740963 1 set Reducing Agent TCEP 740395 107 107 mg NucleoSpin Filters 740606 50 Collection Tubes 2 mL 740600 1000 Visit www mn net com for more detailed product information MACHEREY NAGEL 09 2015 Rev 09 31 RNA isolation 6 3 References Fleige S Pfaffl MW RNA integrity and the effect on the real time qRT PCR performance Mol Aspects Med 2006 Apr Jun 27 2 3 126 39 Epub 2006 Feb 15 Review Imbeaud S Graudens E Boulanger V Barlet X Zaborski P Eveno E Mueller O Schroeder A Auffray C Towards standardization of RNA quality assessment using user independent classifiers of microcapillary electrophoresis traces Nucleic Acids Res 2005 Mar 30 33 6 e56 Miller CL Diglisic S Leister F Webster M Yolken RH Evaluating RNA status for RT PCR in extracts of postmortem human brain tissue Biotechniques 2004 Apr 36 4 628 33 Schoor O Weinschenk T Hennenlotter J Corvin S Stenzl A Rammensee HG Stevanovic S Moderate degradation does not preclude microarray analysis of small amounts of RNA Biotechniques 2003 Dec 35 6 1192 6 1198 201 6 4 Product use restriction warranty NucleoSpin RNA XS kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly described in original MACHEREY
3. 2015 Rev 09 RNA isolation 6 Appendix 6 1 Troubleshooting Problem RNA is degraded no RNA obtained Possible cause and suggestions 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 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 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 rDNase 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 5mM Tris pH 8 5 as d
4. 5 30 uL 35 min 6 preps 110 ug Table 2 Overview on average yields of RNA isolation using NucleoSpin RNA XS Sample Average yield 10 HeLa cells 104 HeLa cells 10 HeLa cells 102 HeLa cells 5 mg mouse kidney 1 mg mouse kidney 1000 1500 ng 100 150 ng 10 15 ng 0 1 1 5 ng 5 8 ug 2 ug 8 MACHEREY NAGEL 09 2015 Rev 09 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 TCEP 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 TCEP should be thawed slowly before starting with the isolation of RNA Wear gloves at all times during the preparation Change gloves frequently REE TEZ are collected by centrifugation and directly lysed by adding Buffer RA1 according to step 2 of the standard protocol see section 5 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 incompl
5. RNA Not necessary 4 Filtrate lysate Not necessary 5 Adjust RNA binding condition Add 1 vol Add one volume of premix to the sample e g premix to 100 uL premix to a 100 uL sample and mix 2x5 s If sample necessary spin down briefly approx 1 s 1000 x g to clear the lid Mix 6 Bind RNA For each preparation take one NucleoSpin RNA XS Column light blue ring placed in a Collection Tube ___ Load iysate and load the lysate to the column Centrifuge for 30s amp y at 11 000 x g a For samples gt 300 uL load in two steps Place the column in a new Collection Tube 2 mL 5 1 re g For high demanding applications the recovery rate can be increased as follows Centrifuge 30 s at 2 000 x g prior to centrifugation for 30 s at 11 000 x g MACHEREY NAGEL 09 2015 Rev 09 23 NucleoSpin RNA XS 7 Desalt silica membrane Not necessary 8 Digest DNA Not necessary 9 Wash and dry silica membrane Add 400 pL Buffer RA3 to the NucleoSpin RNA XS Column Centrifuge for 30 s at 11 000 x g Discard flow 400 pL RA3 through and place the column back into the Collection 11 000 x g Tube F 30 s Add 200 pL Buffer RA3 to the NucleoSpin RNA XS Column Centrifuge for 2 min at 11 000 x g to dry the es 200 pL RA3 membrane Place the column into a nuclease free 11 000 Collection Tube 1 5 mL supplied nd 2 min If for any reason the liquid level in the Collection Tube has r
6. 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 warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish to get an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL ma
7. materials 9 2 4 Elution procedures 10 2 5 Stability of isolated RNA 10 Storage conditions and preparation of working solutions 11 4 Safety instructions 13 5 Protocols 16 5 1 RNA purification from cultured cells laser captured cells or microdissected cryosections 16 5 2 RNA purification from tissue 19 5 3 Clean up and concentration of RNA 23 5 4 rDNase digestion in the eluate 25 6 Appendix 27 6 1 Troubleshooting 27 6 2 Ordering information 31 6 3 References 32 6 4 Product use restriction warranty 32 MACHEREY NAGEL 09 2015 Rev 09 3 RNA isolation 1 Components 1 1 Kit contents NucleoSpin RNA XS 10 preps 50 preps 250 preps REF 740902 10 740902 50 740902 250 Lysis Buffer RA1 6 mL 25 mL 125 mL Wash Buffer RA2 2x1 mL 15 mL 2x15mL Wash Buffer RA3 6mL 12 mL 50 mL Concentrate Membrane Desalting Buffer 10 mL 10 mL 50 mL MDB Reaction Buffer for rDNase 7mL 7mL 30 mL rDNase RNase free 1 vial 1 vial 2 vials Iyphilized size A size C size D Carrier RNA 300 ug 300 ug 300 ug Reducing Agent TCEP 14 mg 3x 14 mg 2x107 mg RNase free H O 13 mL 13 mL 13 mL NucleoSpin Filters 10 50 250 violet rings NucleoSpin RNA XS Columns 10 50 250 light blue rings plus Collection Tubes Collection Tubes 2 mL 30 150 750 Collection Tubes 1 5 mL 10 50 250 User manual 1 1 1 For preparation of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 09 2015 Rev 09 RNA iso
8. of samples to be processed with NucleoSpin RNA XS these disruption methods are often not suitable Add TCEP optional before or after freezing MACHEREY NAGEL 09 2015 Rev 09 9 RNA isolation Recommended disruption and homogenization methods The simple addition of Iysis buffer and subsequent vortexing is usually sufficient to disrupt and homogenize for example up to 10 cultured cells laser captured cells or microdissected cryosections Tissue can be homogenized using a rotor stator homogenizer The spinning rotor disrupts and simultaneousiy homogenizes the sample which is submerged in Iysis buffer by shearing within seconds up to minutes homogenization time depends on sample Keep the rotor tip submerged to avoid excess foaming Select a suitably sized homogenizer 5 7 mm diameter rotors can be used for homogenization in microcentrifuge tubes Bead milling disrupts the tissue samples submerged in Iysis buffer by rapid agitation in the presence of beads Suitable disruption parameters type size and number of beads tube type speed and time of agitation have to be determined empirically for each application 2 4 Elution procedures A high RNA concentration in the elution fraction is desirable for all typical downstream applications In particular with regard to limited volumes of reaction mixtures high RNA concentration can be a crucial criterion Due to a high default elution volume standard kits often re
9. Check that 70 ethanol is available as additional solution in the lab to adjust RNA binding conditions in the Buffer RA1 lysate Check that 96 100 ethanol is available necessary for clean up protocol only Before starting with any NucleoSpin RNA XS protocol prepare the following rDNase Add indicated volume see following table or label on the rDNase vial of RNase free H O 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 Reducing Agent TCEP Add indicated volume of RNase free H O to the TCEP vial and incubate for several minutes at room temperature Mix the vial to completely dissolve the TCEP Store dissolved TCEP at 20 C Carrier RNA Prepare a stock solution before first time using Dissolve the Carrier RNA in 750 uL Buffer RA1 to obtain a 400 ng uL stock solution Prepare a working solution before RNA extraction Dilute 1 100 with Buffer RA1 e g 1 uL Carrier RNA stock solution 99 uL Buffer RA1 to obtain the working solution of 4 ng L Add 5 uL of this working solution 20 ng to every lysate protocol step 3 in section 5 Store stock solution at 20 C do not store working solution prepare it freshly immediately bef
10. Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden GHS Hazard Precaution Component Hazard contents symbol phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze MDB Guanidinium thiocyanate 226 210 233 ethanol 5 20 lt 370 378 Guanidinthiocyanat 1 15 4034235 Ethanol 5 20 WARNING ACHTUNG CAS 593 84 0 64 17 5d RA1 Guanidinium thiocyanate 302 412 260 273 30 60 EUH031 301 312 330 Guanidinthiocyanat 30 60 WARNING CAS 593 84 0 ACHTUNG RA2 Guanidinium thiocyanate 226 302 210 233 260 30 60 ethanol 412 273 301 312 20 35 EUH031 330 370 378 Guanidinhydrochlorid 24 36 WARNING 403 235 Ethanol 20 35 ACHTUNG CAS 593 84 0 64 17 5 rDNase rDNase 90 100 317 334 261 280 rDNase 90 100 lt gt 302 352 304 340 CAS 9003 98 9 DANGER 3334313 GEFAHR 342 311 363 TCEP tris 2 carboxyethyl 315 319 264 280 phosphine hydrochloride 302 352 TCEP HCl 70 100 305 351 338 Tris 2 carboxyethyl WARNING 332 313 phosphinhydrochlorid ACHTUNG 337 313 TCEP HCI 70 100 CAS 51805 45 9 Hazard phrases H226 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar H302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken MACHEREY NAGEL 09 2015 Rev 09 13 RNA isolation H315 H317 H319 H334 H412 Causes skin irritation Verursacht Hautreizungen May cause an a
11. 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 The respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for IN VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for IN VITRO diagnostic use Please pay attention to the package of the product IN VITRO diagnostic products are expressly marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR IN VITRO DIAGNOSTIC USE ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE 32 MACHEREY NAGEL 09 2015 Rev 09 RNA isolation No claim or representations is intended for its use to identify any specific organism or for clinical use included but not
12. UHRUNG MIT DER HAUT Mit viel Wasser waschen P304 340 IF INHALED Remove person to fresh air and keep comfortable for breathing BEI EINATMEN Die Person an die frische Luft bringen und fur ungehinderte Atmung sorgen P305 351 338 IF IN EYES Rinse cautiously with water for several minuts Remove contact lenses if present and easy to do Continue rinsing BEI KONTAKT MIT DEN AUGEN Einige Minuten lang behutsam mit Wasser aussp len Eventuell vorhandene Kontaktlinsen nach M glichkeit entfernen Weiter aussp len P330 Rinse mouth Mund aussp len P332 313 If skin irritation occurs Get medical advice attention Bei Hautreizung Arztlichen Rat einholen arztliche Hilfe hinzuziehen P333 313 If skin irritation or rash occurs Get medical advice attention Bei Hautreizung oder ausschlag rztlichen Rat einholen rztliche Hilfe hinzuziehen 14 MACHEREY NAGEL 09 2015 Rev 09 RNA isolation P 337 313 P 342 311 P363 P370 378 P403 235 If eye irritation persists Get medical advice attention Bei anhaltender Augenreizung rztlichen Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTER doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM Arzt anrufen Wash contaminated clothing before reuse Kontaminierte Kleidung vor erneutem Tragen waschen In case of fire Use to extinguish Bei Brand zum L schen verwenden Store in a well ventilated plac
13. a membrane Add 100 uL MDB Membrane Desalting Buffer and centrifuge at 11 000 x g for 30 s to dry the membrane It is not necessary to use a fresh Collection Tube after this centrifugation step 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 rDNase reaction mixture in a sterile microcentrifuge tube not provided for each isolation add 3 uL reconstituted rDNase also see section 3 to 27 uL Reaction Buffer for rDNase Mix by flicking the tube Apply 25 pL rDNase reaction mixture directly onto the center of the silica membrane of the column Close the lid Incubate at room temperature for 15 min It is not necessary to use a new Collection Tube after the incubation step 100 pL 70 EtOH Mix Load Iysate 11 000 x g 30 s 100 pL MDB 11 000 x g 30 s 25 uL rDNase reaction mixture RT 15 min MACHEREY NAGEL 09 2015 Rev 09 17 NucleoSpin RNA XS 9 Wash and dry silica membrane Add 100 uL Buffer RA2 to the NucleoSpin RNA XS Column Incubate for 2 min at RT Centrifuge for 30 s at 11 000 x g 100 pL RA2 Place the column into a new Collection Tube 2 mL RT Buffer RA2 will inactivate the rDNase 2 min 30s Add 400 pL Buffer RA3 to the NucleoSpin RNA XS Colum
14. centrifugation discard flow through and centrifuge again 25 pL rDNase reaction mixture RT 15 min 100 pL RA2 RT 2 min 11 000 x g 30s 400 pL RA3 11 000 x g 30s 200 pL RA3 11 000 x g 2 min MACHEREY NAGEL 09 2015 Rev 09 21 NucleoSpin RNA XS 10 Elute highly pure RNA Elute the RNA in 10 pL H O RNase free supplied and 10 pL centrifuge at 11 000 x g for 30 s RNase free If higher RNA concentrations or higher elution volumes H20 are desired elution volume may be varied in the range of 5 30 uL Au For further details on alternative elution procedures see section 2 4 22 MACHEREY NAGEL 09 2015 Rev 09 NucleoSpin RNA XS 5 3 Clean up and concentration of RNA Before starting the preparation Check if Wash Buffer RA3 were prepared according to section 3 1 Supply sample Provide up to 300 uL sample such as prepurified RNA e g phenol purified or RNA from reaction mixtures e g labelling reactions in a microcentrifuge tube not I Sample provided For appropriate sample amounts see section 2 2 2 Prepare lysis binding buffer premix 25 uL RA1 For every 100 uL of sample combine 25 uL Buffer RA1 73 ar FION with 75 uL ethanol 96 100 and mix 96 100 per 100 uL If processing multiple samples the preparation of a sample master premix 1 volume Buffer RA1 plus 3 volumes ethanol 96 100 is recommended Mix 3 Add Carrier
15. e Keep cool An einem gut bel fteten Ort aufbewahren K hl halten For further information please see Material Safety Data Sheets www mn net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenbl ttern www mn net com AS The symbol shown on labels refers to further safety information in this section Das auf Etiketten dargestellte Symbol weist auf weitere Sicherheitsinformationen dieses Kapitels hin MACHEREY NAGEL 09 2015 Rev 09 15 NucleoSpin RNA XS 5 Protocols 5 1 RNA purification from cultured cells laser captured cells or microdissected cryosections Before starting the preparation Check if TCEP Carrier RNA rDNase and Wash Buffer RA3 were prepared according to section 3 1 Supply sample Provide sample such as a pellet of up to 10 cultured cells laser captured cells or microdissected cryosections in a microcentrifuge tube not provided For appropriate sample amounts see section 2 2 2 Lyse and homogenize cells Add 100 pL Buffer RA1 and 2 uL TCEP to the cell sample and vortex vigorously 2 x 5 s If multiple samples are processed the preparation of a master premix is recommended e g 1 1 mL 100 pL Buffer RA1 and 22 uL TCEP for 10 preparations Use y RA1 102 uL of the premix 2 pL TCEP This procedure is usually sufficient to homogenize cultured cells laser captured cells or microdissected cryosections For further comments on homogenization methods s
16. eached the NucleoSpin RNA XS Column after centrifugation discard flow through and centrifuge again 10 Elute highly pure RNA Elute the RNA in 10 pL H O RNase free supplied and centrifuge at 11 000 x g for 30 s 10 pL If higher RNA concentrations or higher elution volumes RNase free are desired elution volume may be varied in the range H20 of 5 30 uL 11 000 x g For further details on alternative elution procedures see 30 s section 2 4 24 MACHEREY NAGEL 09 2015 Rev 09 NucleoSpin RNA XS 5 4 rDNase digestion in the eluate The on column rDNase digestion in the standard protocol is very efficient and thus results in minimal residual DNA This DNA will not be detectable in most downstream applications However removal of DNAto 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 contaminating 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 lt 200 bp DNA digestion in solution can efficiently destroy contaminating DNA However
17. ee section 2 3 3 Add Carrier RNA This step may be skipped when working with small amounts of sample for example less than 10 cells Add 5 uL Carrier RNA working solution 20 ng to 5uL the Iysate Mix by vortexing 2 x5 s Spin down briefly Carrier RNA approx 1 s 1000 x g to clear the lid ui ix For preparation of Carrier RNA working solution see section 3 4 Filtrate lysate optional Place a NucleoSpin Filter violet ring in a Collection Tube 2 mL supplied apply the mixture and centrifuge 11 000 x g for 30 s at 11 000 x g 30 s 5 16 MACHEREY NAGEL 09 2015 Rev 09 NucleoSpin RNA XS Adjust RNA binding condition Discard the NucleoSpin Filter violet ring Add 100 uL ethanol 70 to the homogenized Iysate and mix by pipetting up and down 5 times Alternatively add 100 uL ethanol 70 to the sample ina 1 5 mL microcentrifuge tube not provided and mix by vortexing 2x 5s Spin down briefly approx 1s 1000 x g to clear the lid Pipette lysate up and down two times before loading the lysate Bind RNA For each preparation take one NucleoSpin RNA XS Column light blue ring placed in a Collection Tube Load the lysate to the column Centrifuge for 30s at 11 000 x g Place the column in a new Collection Tube 2 mL The maximum loading capacity of NucleoSpin RNA XS Columns is 600 uL Repeat the procedure if larger volumes are to be processed Desalt silic
18. ent in virtually all biological 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 temperature The eluate however should be treated 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 2 2 Kit specifications The NucleoSpin RNA XS kit is recommended for the isolation of RNA from very small samples Typical sample material comprises small amounts of cells up to 1 x 10 and tissue up to 5 mg such as pellets of cultured cells laser captured cells microdissected cryosections biopsy samples fine needle aspirates and flow cytometer sorted cells Table 1 page 7 e The innovative column design with a funnel shaped thrust ring and a small silica membrane area allows elution of RNA in as little as 5 30 uL Thus high
19. ete removal of the cell culture medium in order to 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 LEREM are often tough and should be disrupted mechanically to be available for lysis 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 Thawing of undisrupted animal tissue should only be done in the presence of Buffer RA1 under simultaneous mechanical disruption for example with a rotor stator homogenizer or a bead mill This ensures that the RNA is not degraded by RNases before the preparation has started Commonly used techniques for disruption of animal tissues are for example grinding with pestle and mortar or using a syringe and needle for multiple passage of the sample through the needle However due to the small size
20. forming absorption measurements close to the detection limit of the photometer the measurement may be influenced by minor amounts of silica abrasion In order to prevent incorrect Asgo quantification of small RNA amounts centrifuge the eluate for 30s at gt 11 000 x g and take an aliquot for measurement without disturbing any sediment Alternatively use a silica abrasion insensitive RNA quantification method e g RiboGreen fluorescent dye Unexpected A260 A2g0 ratio Measurement not in the range of photometer detection limit In order to obtain a significant Azgo Azgo ratio it is necessary that the initially measured Ago and Aggo values are significantly above the detection limit of the photometer used An Azgo value close to the background noise of the photometer will cause unexpected Axgo Azgo ratios 30 MACHEREY NAGEL 09 2015 Rev 09 RNA isolation 6 2 Ordering information Product REF Pack of NucleoSpin RNA XS 740902 10 50 250 10 50 250 NucleoSpin RNA Clean up XS 740903 10 50 250 10 50 250 NucleoSpin totalRNA FFPE XS 740969 10 50 250 10 50 250 NucleoSpin RNA 740955 10 50 250 10 50 250 NucleoZOL 740404 200 200 mL NucleoSpin RNA Blood 740200 10 50 10 50 NucleoSpin totalRNA FFPE 740982 10 50 250 10 50 250 NucleoSpin RNA Midi 740962 20 20 NucleoSpin RNA Protein 740933 10 50 250 10 50 250 NucleoSpin TriPrep 740966 10 50 250 10 50 250 NucleoSpin RNA Clean up 740948 10 50 250
21. iluent Please see also Manchester K L 1995 Value of Aggo Aago 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 MACHEREY NAGEL 09 2015 Rev 09 27 RNA isolation Problem Possible cause and suggestions 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 Poor RNA allow tissues to thaw before addition of Buffer RA1 Perform quality or yield disruption of samples immediately after addition of Lysis continued Buffer RA1 Insufficient disruption and or homogenization of starting material Ensure thorough sample disruption and use NucleoSpin Filters for easy homogenization of disrupted starting material Carry over of guanidinium thiocyanate Carefully load the lysate to the NucleoSpin RNA Il Column Low A A and try to avoid a contamination of the upper part of the aun See LESI column and the column lid ratio Make sure that residual Wash Buffer RA2 is washed away with Wash Buffer RA3 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 ove
22. kes 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 whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a pa
23. l performance with smallest samples It is recommended adding Carrier RNA to the sample lysate 20 ng per sample Such small amounts typically do not interfere with subsequent RT PCR even in oligo dT primed reverse transcriptions The small amount of Carrier RNA transfered into a reverse transcription reaction is commonly not significantly influencing the outcome of the reaction due tothe large excess of oligo dT primer The benefit of adding Carrier RNA to the sample lysate depends on sample type amount and kind of downstream RNA analysis Carrier RNA should be omitted when subsequent to RNA isolation a poly A RNA isolation is performed RNA sequencing is performed rDNase is supplied in the kit DNA contaminations are removed by on column digestion with rDNase For most demanding applications e g expression analysis of plasmid transfected cells plastidial or mitochondrial genes a subsequent digestion with rDNase in the eluate is possible MACHEREY NAGEL 09 2015 Rev 09 7 RNA isolation Table 1 Kit specifications at a glance Parameter NucleoSpin RNA XS Format Sample material Fragment size Typical yield Azgo ago Typical RIN RNA integrity number Elution volume Preparation time Binding capacity Mini spin column XS design Small amounts of tissue lt 5 mg tissue lt 100 000 cultured cells gt 200 nt See table 2 for examples 1 9 2 1 gt 9 depending on sample quality
24. lation 1 2 Reagents consumables and equipment to be supplied by user Reagents e 96 100 ethanol to prepare Wash Buffer RA3 and for the clean up procedure section 5 3 70 ethanol to adjust RNA binding condition Consumables 1 5 mL microcentrifuge tubes Sterile RNase free tips Equipment e Manual pipettors Centrifuge for microcentrifuge tubes Vortex mixer Personal protection equipment 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 XS kit is used for the first time Experienced users however may refer to the Protocol at a glance instead The Protocol at a glance is designed to be used only as a supplemental tool for quick referencing while performing the purification procedure All technical literature is available on the internet at www mn net com Please contact Technical Service regarding information about changes of the current user manual compared to previous revisions MACHEREY NAGEL 09 2015 Rev 09 5 RNA isolation 2 Product description 2 1 The basic principle One of the most important aspects isolating 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 pres
25. leoSpin Filter violet ring add 200 pL ethanol 70 to the homogenized Iysate and mix by pipetting up and down 5 times Alternatively transfer flow through into a new 1 5 mL microcentrifuge tube not provided add 200 pL ethanol 70 and mix by vortexing 2 x 5 s Spin down briefly approx 1 s 1000 x g to clear the lid Pipette lysate up and down two times before loading the lysate 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 6 Do not centrifuge 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 XS Column light blue ring placed in a Collection Tube and load the lysate to the column Centrifuge for 30 s at 11 000 x g Place the column in a new Collection Tube 2 mL The maximum loading capacity of NucleoSpin RNA XS Columns is 600 uL Repeat the procedure if larger volumes are to be processed Desalt silica membrane Add 100 uL MDB Membrane Desalting Buffer and centrifuge at 11 000 x g for 30 s to dry the membrane It is not necessary to use a fresh Collection Tube after this centrifugation step Salt removal will make the following rDNase digest much more effective If the column outlet has come into contact with the flow th
26. llergic skin reaction Kann allergische Hautreaktionen verursachen Causes serious eye irritation Verursacht schwere Augenreizung May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursachen Harmful to aquatic life with long lasting effects Sch dlich f r Wasserorganismen mit langfristiger Wirkung Precaution phrases P 210 Keep away from heat hot surfaces sparks open flames and other ignition sources No smoking Von Hitze heissen Oberfl chen Funken offenen Flammen sowie anderen Z ndquellenarten fernhalten Nicht rauchen P233 Keep container tightly closed Beh lter dicht verschlossen halten P260 Do not breathe dust fume gas mist vapours spray Staub Rauch Gas Nebel Dampf Aerosol nicht einatmen P261 Avoid breathing dust fume gas mist vapours spray Einatmen von Staub Rauch Gas Nebel Dampf Aerosol vermeiden P264 Wash thoroughly after handling Nach Handhabung gr ndlich waschen P273 Avoid release to the environment Freisetzung in die Umwelt vermeiden P280 Wear protective gloves protective clothing eye protection face protection Schutzhandschuhe Schutzkleidung Augenschutz Gesichtsschutz tragen P301 312 IF SWALLOWED Call a POISON CENTER doctor if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen P302 352 IF ON SKIN Wash with plenty of water BEI BER
27. ly concentrated RNA is eluted ready for common downstream applications e g RT PCR The RNA yield strongly depends on the sample type quality and amount see Table 2 page 8 for details High quality RNA RNA Integrity Number RIN gt 9 according to Agilent 2100 Bioanalyzer assays can be obtained from small samples e g 10 cells 0 1 mg tissue as well as from larger samples 10 cells 5 mg tissue rRNA ratios 28S 18S of 1 8 2 0 can be obtained Since RNA quality always depends on the sample quality see section 6 3 for further aspects The NucleoSpin RNA XS kit allows purification of RNA with an Asgo Aago ratio generally exceeding 1 9 measured in TE buffer pH 7 5 Due to the high RNA purity large amounts of eluates can be used as template in RT PCR without inhibition e g 8 uL of 10 uL eluates as template in a 20 uL qRT PCR setup generating stronger signal compared to reactions with less template in a LightCycler PCR with the Sigma SYBR Green Quantitative RT PCR Kit 6 MACHEREY NAGEL 09 2015 Rev 09 RNA isolation The preparation time is approximately 45 min for 12 samples As Reducing Agent TCEP Tris 2 carboxyethyl phosphine is supplied in the kit TCEP is odorless more stable more specific for disulfide bonds and less toxic than other commonly used reducing agents Carrier RNA poly A RNA poly A potassium salt prepared from ADP with polynucleotide phosphorylase is included for optima
28. n Centrifuge for 30 s at 11 000 x g Discard flow through and place the column back into the Collection amp Tube 400 uL Note Make sure that residual buffer from the previous RA3 steps is washed away with Buffer RA3 especially if 11 000 x g the lysate has been in contact with the inner rim of the 30 s column during loading of the lysate onto the column For efficient washing of the inner rim flush it with Buffer RA3 Eo NN Add 200 uL Buffer RA3 to the NucleoSpin RNA XS RA3 Column Centrifuge for 2 min at 11 000 x g to dry the membrane Place the column into a nuclease free 11 000 x g Collection Tube 1 5 mL supplied 2 min If for any reason the liquid level in the Collection Tube has reached the NucleoSpin RNA XS Column after centrifugation discard flow through and centrifuge again 10 Elute highly pure RNA Elute the RNA in 10 L H O RNase free supplied and 10 pL centrifuge at 11 000 x g for 30 s da i If higher RNA concentrations or higher elution volumes are desired elution volume may be varied in the range For further details on alternative elution procedures see 30 s section 2 4 18 MACHEREY NAGEL 09 2015 Rev 09 NucleoSpin RNA XS 5 2 RNA purification from tissue Before starting the preparation Check if TCEP Carrier RNA rDNase and Wash Buffer RA3 were prepared according to section 3 1 Supply sample Provide tissue sample such as a biopsy in a microcentrifuge t
29. ore use Wash Buffer RA3 Add the indicated volume of 96 100 ethanol to Wash 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 Due to the production procedure lyophilized Carrier RNA might hardly be visible in the vial MACHEREY NAGEL 09 2015 Rev 09 11 RNA isolation NucleoSpin RNA XS 10 preps 50 preps 250 preps REF 740902 10 740902 50 740902 250 Wash Buffer RA3 6 mL 12 mL 50 mL Concentrate Add 24 mL ethanol Add 48 mL ethanol Add 200 mL ethanol to each bottle rDNase 1 vial size A 1 vial size C 2 vials size D RNase free Add 55 uL Add 230 uL Add 540 uL lyophilized RNase free H O RNase free H O RNase free H O to each vial Carrier RNA 300 ug 300 ug 300 ug Add 750 uL Buffer RA1 to obtain concentrated stock solution Dilute 1 100 with Buffer RA1 to obtain working solution Reducing Agent 14mg 3x 14mg 2x 107mg TCEP Add 100 uL Add 100 uL Add 750 uL RNase free H O RNase free H O RNase free H O to each vial to each vial 12 MACHEREY NAGEL 09 2015 Rev 09 RNA isolation 4 Safety instructions The following components of the NucleoSpin RNA XS kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features do not need to be labeled with H and P phrases up to 125 mL or 125 g
30. ort protocol 5 4 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 e Check if Buffer RA3 has been equilibrated to room temperature before use Washing at lower temperatures lowers efficiency of salt removal by Buffer RAS Depending on the robustness of the used RT PCR system RT PCR might be inhibited if complete eluates are used as template for RT PCR Use less eluate as template Store isolated RNA properly Eluted RNA should always be kept on ice for optimal stability since trace contaminations of omnipresent RNases general lab ware fingerprints dust will degrade the isolated RNA For short term storage freeze at 20 C for long term storage freeze at 70 C MACHEREY NAGEL 09 2015 Rev 09 29 RNA isolation Problem Discrepancy between Ago quantification values and PCR quantification values Possible cause and suggestions Silica abrasion from the membrane Due to the typically low RNA content in very small samples and the resulting low total amount of isolated RNA an RNA quantification via As absorption measurement is often hampered due to the low sensitivity of the absorption measurement When per
31. rall yield Reduce amount of sample material Column or use larger volume of Buffer RA1 Poor RNA Av n al Insufficient disruption and or homogenization of starting or yield material Ensure thorough sample disruption and use NucleoSpin Filters for easy homogenization of disrupted starting material Contamination of RNA with genomic DNA rDNase not active Reconstitute and store Iyophilized rDNase according to instructions given in section 3 DNase solution not properly applied Pipette rDNase solution directly onto the center of the silica membrane and close the lid Too much cell material used Reduce quantity of cells or tissue used 28 MACHEREY NAGEL 09 2015 Rev 09 RNA isolation Problem Contamination of RNA with genomic DNA continued Possible cause and suggestions 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 probability of DNA detection with PCR increases with the number of DNA copies per preparation single copy target lt plastidial mitochondrial target lt 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 supp
32. rough for any reason discard the flow through and centrifuge again for 30 s at 11 000 x g 200 pL y 70 EtOH Mix Load lysate ed 11 000 x g 30s 100 pL MDB cS 11 000 x g 30s 20 MACHEREY NAGEL 09 2015 Rev 09 NucleoSpin RNA XS 8 Digest DNA Prepare rDNase reaction mixture in a sterile microcentrifuge tube not provided for each isolation add 3 uL reconstituted rDNase also see section 3 to 27 uL Reaction Buffer for rDNase Mix by flicking the tube Apply 25 pL rDNase reaction mixture directly onto the center of the silica membrane of the column Close the lid Incubate at room temperature for 15 min It is not necessary to use a new Collection Tube after the incubation step Wash and dry silica membrane Add 100 pL Buffer RA2 to the NucleoSpin RNA XS Column Incubate for 2 min at RT Centrifuge for 30 s at 11 000 x g Place the column into a new Collection Tube 2 mL Buffer RA2 will inactivate the rDNase Add 400 pL Buffer RA3 to the NucleoSpin RNA XS Column Centrifuge for 30 s at 11 000 x g Discard flow through and place the column back into the Collection Tube Add 200 pL Buffer RA3 to the NucleoSpin RNA XS Column Centrifuge for 2 min at 11 000 x g to dry the membrane Place the column into a nuclease free Collection Tube 1 5 mL supplied If for any reason the liquid level in the Collection Tube has reached the NucleoSpin RNA XS Column after
33. rt 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 MACHEREY NAGEL 09 2015 Rev 09 33 RNA isolation may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications Last updated 07 2010 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 24 21 969 270 tech bio mn net com 34 MACHEREY NAGEL 09 2015 Rev 09 MACHEREY NAGEL Germany and international Tel 49 24 21 969 0 E mail info mn net com MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 52355 D ren Germany Switzerland France USA MACHEREY NAGEL AG MACHEREY NAGEL EURL MACHEREY NAGEL Inc Tel 41 62 388 55 00 Tel 33 388 68 22 68 Tel 1 484 821 0984 E mail sales ch mn net com E mail sales fr mn net com E mail sales us mn net com A034939 0951 5
34. 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 recombinant RNase free DNase rDNase inthe NucleoSpin RNA XS kits facilitates such a digestion in solution in order to remove even traces of contaminating DNA A Digest DNA Reaction setup Prepare enzyme buffer premix Add 1 uL rDNase to 10 uL Reaction Buffer for rDNase Add 1 10 volume of enzyme buffer premix to the eluted RNA e g to 10 uL RNA add 1 uL of the premix comprising buffer and enzyme B Incubate sample Incubate for 10 min at 37 C MACHEREY NAGEL 09 2015 Rev 09 25 RNA isolation Repurify RNA Repurify RNA with a suitable RNA cleanup procedure for example following section 5 3 by ethanol precipitation or with the NucleoSpin RNA Clean up XS kit see ordering information 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 respectively 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 max speed Wash RNA pellet with 70 ethanol Dry RNA pellet and resuspend RNA in RNase free H O 26 MACHEREY NAGEL 09
35. sult in weakly concentrated RNA if only small samples are processed Such RNA often even requires a subsequent concentration to be suitable for the desired application In contrast to standard kits NucleoSpin RNA XS allows an efficient elution in a very small volume resulting in highly concentrated RNA Elution volumes in the range of 5 30 uL are recommended the default volume is 10 uL 2 5 Stability of isolated RNA Eluted RNA should immediately be put and always kept on ice during work for optimal stability Contamination with almost omnipresent RNases general lab ware fingerprints dust may be a risk for isolated RNA For short term storage freeze at 20 C for long term storage freeze at 70 C 10 MACHEREY NAGEL 09 2015 Rev 09 RNA isolation 3 Storage conditions and preparation of working solutions Attention Buffers RA1 RA2 and MDB contain chaotropic salt and detergents Wear gloves and goggles CAUTION Buffers RA1 RAZ and MDB contain guanidinium thiocyanate 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 Reducing Agent TCEP and Carrier RNA 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 precipitation of salts
36. ube not provided For appropriate sample amounts see section 2 2 2 Lyse and homogenize tissue Add 200 uL Buffer RA1 and 4 uL TCEP to the tissue sample and vortex vigorously 2 x 5 s Disruption with a rotor stator homogenizer or with a shaker and steel balls are recommended methods for the homogenization of tissue samples For further comments on homogenization methods see section 2 3 If multiple samples are processed the preparation e of a master premix is recommended e g 2 2 mL Buffer RA1 and 44 uL TCEP for 10 preparations Use 204 uL of the premix 200 pL RA1 4 uL TCEP 38 Add Carrier RNA Add 5 L Carrier RNA working solution 20 ng to 5uL the lysate Mix by vortexing 2 x 5 s Spin down briefly Carrier RNA approx 1 s 1000 x g to clear the lid i ix For preparation of Carrier RNA working solution see section 3 4 Filtrate lysate Reduce viscosity and clear the lysate by filtration through NucleoSpin Filter violet ring Place the NucleoSpin Filter violet ring in a Collection Tube H 11 000 x g 2 mL provided apply the mixture and centrifuge for i 30s 30 s at 11 000 x g amp In case of visible pellet formation depending on sample amount and nature transfer supernatant without any formed pellet to anew 1 5 mL microcentrifuge tube not included MACHEREY NAGEL 09 2015 Rev 09 19 NucleoSpin RNA XS Adjust RNA binding condition Discard the Nuc
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