Total RNA Isolation
Contents
1. 11 000 x g 30s 100 jil 70 EtOH Mix Load lysate 11 000 x g 30s 100 ul MDB 11 000x g 30s 16 MACHEREY NAGEL 01 2010 Rev 04 NucleoSpin RNA XS Digest DNA Prepare rDNase reaction mixture in a sterile micro 25 ul centrifuge tube not provided for each isolation add DN H 3 ul reconstituted rDNase also see section 3 to 27 pl psdse Reaction Buffer for rDNase Mix by flicking the tube reaction mixture Apply 25 ul rDNase reaction mixture directly onto the center of the silica membrane of the column Close the RT lid Incubate at room temperature for 15 min 15 min It is not necessary to use a new Collection Tube after the incubation step Wash and dry silica membrane 1 wash 100 jil RA2 Add 100 pl Buffer RA2 to the NucleoSpin RNA XS RT Column Incubate for 2 min at RT Centrifuge for 30 s at 2 min 11 000 x g Place the column into a new Collection Tube 2 ml ien g Buffer RA2 will inactivate the rDNase Add 400 pl Buffer RA3 to the NucleoSpin RNA XS 400 pl RA3 Column Centrifuge for 30 s at 11 000 x g Discard flow through and place the column back into the Collection 11 000 x g Tube 30s Add 200 yl Buffer RA3 to the NucleoSpin RNA XS Column Centrifuge for 2 min at 11 000 x g to dry the 200 pl RAS membrane Place the column into a nuclease free Collection Tube 1 5 ml supplied 11 000 x g 2 min If for any reason the liquid level in the Collection Tube has reac2. MACHEREY NAGEL 01 2010 Rev 04 11 Total RNA Isolation NucleoSpin RNA XS 10 preps 50 preps 250 preps Cat No 740902 10 740902 50 740902 250 Wash Buffer RA3 2 ml 7 ml 2x20ml Concentrate Add 8 ml ethanol Add 28 ml ethanol Add 80 ml ethanol to each bottle rDNase 1 vial size A 1 vial size C 2 vials size D RNase free Add 55 yl Add 230 yl Add 540 yl 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 yl Buffer RA1 to obtain concentrated stock solution Dilute 1 100 with Buffer RA1 to obtain working solution Reducing Agent 14 mg 3x14 mg 2x 107 mg 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 01 2010 Rev 04 Total RNA Isolation 4 Safety instructions risk and safety phrases 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 Component Hazard Hazard Risk Safety contents symbol phrases phrases rDNase rDNase x Xi May cause sensiti R 42 43 S 22 24 RNase free lyophillized zation by inhalation and skin contact RA1 Guanidine x Xn Harmful by inhala R 20 21 22 8 18 thiocyanate tion in contact with skin and if swal lowed RA2 Guanidine x Xn Flammable Harmful R 10 S 13 16 thiocyanate by inhalation in c
3. 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 Add TCEP optional before or after freezing 8 MACHEREY NAGEL 01 2010 Rev 04 Total RNA Isolation 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 prepa ration 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
4. Total RNA Isolation User Manual NucleoSpin RNA XS January 2010 Rev 04 MACHEREY NAGEL MN Total RNA Isolation Protocol at a glance Rev 04 XS NucleoSpin RNA XS 1 Supply sample Use up to 5 x 10 cultured cells or 5 mg tissue samples 2 Lyse and homogenize cells 100 ul RA1 2 ul TCEP Mix 3 Add Carrier RNA 5 ul Carrier RNA working solution Mix 4 Filtrate lysate optional e 11 000 x g 30s 5 Adjust RNA 100 pl 70 ethanol binding condition Mix 6 Bind RNA S Load lysate 5 11 000 x g 2 30s 7 Desalt silica membrane 100 ul MDB e gt 11 000 x g e 30s 8 Digest DNA 25 ul DNase n reaction mixture E RT ku 15 min 9 Wash and dry silica 1 wash 100 pl RA2 membrane RT 2 min 11 000 x g 30s d gt 254 wash 400 jil RA3 11 000 x g 2 min 3 wash 200 pl RA3 11 000 x g 2 min 10 Elute highl RNA 0 ute highly pure 10 ul RNase free H O e gt 11 000 x g 30 s MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 D 52355 D ren Germany Tel 49 0 24 21 969 270 www mn net com e mail tech bio mn net com Total 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 starting
5. Overview on average yields of total RNA isolation using NucleoSpin RNA XS Sample Average yield 10 HeLa cells 1000 1500 ng 10 HeLa cells 100 150 ng 102 HeLa cells 10 15 ng 10 HeLa cells 0 1 1 5 ng 5 mg mouse kidney 5 8 jg 1 mg mouse kidney 2 ug 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 TECP 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 total RNA Wear gloves at all times during the preparation Change gloves frequently e uei REE 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 incomplete removal of the cell culture medium in order to allow full lysis activity of the lysis buffer
6. as described in step 6 Do not centrifuge the ethanolic lysate before loading it onto the col umn 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 cam 2 gt 11 000 x g 30s 200 jil 70 EtOH Mix Load lysate 11 000 x g 30s 20 MACHEREY NAGEL 01 2010 Rev 04 NucleoSpin RNA XS Desalt silica membrane Add 100 gl 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 micro centrifuge tube not provided for each isolation add 3 pl reconstituted rDNase also see section 3 to 27 pl Reaction Buffer for rDNase Mix by flicking the tube Apply 25 ul rDNase reaction mixture directly onto the center of the silica membrane o
7. immediately inactivates RNases which are present 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 RNAis 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 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 total RNA from very small samples Typical sample material comprises small amounts of cells up to 5 x 105 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 allo
8. preparations Use 204 ul of the premix 3 Add Carrier RNA the lysate Mix by vortexing 2 x 5 s Spin down briefly Carrier RNA Add 5ul Carrier RNA working solution 20 ng to 5 pl approx 1 s 1000 x g to clear the lid Mix For preparation of Carrier RNA working solution see section 3 MACHEREY NAGEL 01 2010 Rev 04 19 NucleoSpin RNA XS 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 2 ml provided apply the mixture and centrifuge for 30 s 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 microcentrifuge tube not included Adjust RNA binding condition Discard the NucleoSpin Filter violet ring add 200 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 200 pl ethanol 7096 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
9. section 2 4 MACHEREY NAGEL 01 2010 Rev 04 25 NucleoSpin RNA XS 5 4 Support protocol NucleoSpin RNA XS 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 DNA to a completely undetectable level is challeng ing 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 contami nating genomic DNA Especially if e 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 e the amplicon is relatively small 200 bp DNA digestion in solution can efficiently destroy contaminating DNA However strin gent RNase control and subsequent repurification of the RNA in order to remove buffer salts DNase and digested DNA are usually required Thehigh quality recombinant RNase free DNase rDNase inthe NucleoSpin RNA XS kits facilitates such a digestion in solution in order to remove even traces of contaminat ing DNA A Digest DNA Reaction setup Prepare enzyme buffer premix Ad
10. A 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 care ful 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 com pletely 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 ul 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 before use Wash Buffer RA3 Add the indicated volume of 96 10096 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 20 25 C for up to one year
11. AGEL 01 2010 Rev 04 NucleoSpin RNA XS 5 Protocols 5 1 Total RNA purification from cultured cells laser captured cells or microdissected cryosections with NucleoSpin RNA XS Before starting the preparation e 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 5 x 105 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 pl 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 Buffer RA1 and 22 ul TCEP for 10 preparations Use 102 ul of the premix 100 pl RA1 2 ul TCEP e This procedure is usually sufficient to homogenize cultured cells laser captured cells or microdissected cryosections For further comments on homogenization methods see section 2 3 3 Add Carrier RNA 5 jil Add 5 ul Carrier RNA working solution 20 ng to Carrier RNA the lysate Mix by vortexing 2 x 5 s Spin down briefly approx 1 s 1000 x g to clear the lid e Mix For preparation of Carrier RNA working solution see section 3 MACHEREY NAGEL 01 2010 Rev 04 15 NucleoSpin RNA XS Filtrate lysate optional Place a NucleoSpin Fil
12. d 1 pl rDNase to 10 pl 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 26 MACHEREY NAGEL 01 2010 Rev 04 NucleoSpin RNA XS C 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 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 7096 ethanol Dry RNA pellet and resuspend RNA in RNase free H O MACHEREY NAGEL 01 2010 Rev 04 27 Total RNA Isolation 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions RNA is degraded no RNA obtained HNase 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 durin
13. due to the small size of samples to be processed with NucleoSpin RNA XS these disruption methods are often not suitable Recommended disruption and homogenization methods The simple addition of lysis 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 simultaneously homogenizes the sample which is submerged in lysis 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 microcen trifuge tubes Bead milling disrupts the tissue samples submerged in lysis 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 MACHEREY NAGEL 01 2010 Rev 04 9 Total RNA Isolation 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 result in weakly concentra
14. e detection limit of the photometer used An A value close to the background noise of the photometer will cause unexpected A A ratios 260 7 280 MACHEREY NAGEL 01 2010 Rev 04 31 Total RNA Isolation 6 2 Ordering information Product Cat No 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 RNA II 740955 10 20 50 250 10 20 50 250 NucleoSpin FFPE RNA 740969 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 RNA Clean up 740948 10 50 250 10 50 250 NucleoSpin RNA Buffer Set Buffer RA1 rDNase Set NucleoSpin Filters Collection Tubes 2 ml 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 IN THE USA IS PROHIBITED FOR PATENT REASONS 32 MACHEREY NAGEL 01 2010 Rev 04 Total RNA Isolation 6 3 References Fleige S Pfaffl MW RNA integrity and the effect on the real time qRT PCR perfor mance 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 independe
15. ection Tube and load the lysate to the column Centrifuge for 30 s at Load Weak 11 000 x g oag lysate For samples gt 300 ul load in two steps 11 000 x g Place the column in a new Collection Tube 2 ml 30s 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 7 Desalt silica membrane Not necessary 8 Digest DNA Not necessary 9 Wash and dry silica membrane 400 pl RA3 Add 400 pl Buffer RA3 to the NucleoSpin RNA XS Column Centrifuge for 30 s at 11 000 x g Discard flow 11 000x g through and place the column back into the Collection 30s Tube Add 200 yl Buffer RA3 to the NucleoSpin RNA XS 200 pl 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 centrifuga tion discard flow through and centrifuge again 24 MACHEREY NAGEL 01 2010 Rev 04 NucleoSpin RNA XS 10 Elute highly pure RNA Elute the RNA in 10 pl H O RNase free supplied and 10 ul centrifuge at 11 000 x g for 30 s RNase free H O If higher RNA concentrations or higher elution volumes 2 are desired elution volume may be varied in the range of 5 30 ul 11 000 x g For further details on alternative elution procedures see ane
16. erial used e Reduce quantity of cells or tissue used 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 plastidial mitochondrial target plasmid transfected into cells decreasing of PCR amplicon size e Use larger PCR targets e g 500 bp or intron spanning primers if possible Use support protocol 5 4 for subsequent rDNase diges tion 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 tem perature before use Washing at lower temperatures lowers efficiency of salt removal by Buffer RAS e 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 30 MACHEREY NAGEL 01 2010 Rev 04 Total RNA Isolation Problem Pos
17. es 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 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 mn net com Last updated 12 2006 Rev 02 34 MACHEREY NAGEL 01 2010 Rev 04
18. f 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 yl 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 yl 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 100 ul MDB 11 000x g 30s 25 ul rDNase reaction mixture RT 15 min 100 pl RA2 RT 2 min 11 000 x g 30s 6 400 pl RAS 11 000 x g 30s MACHEREY NAGEL 01 2010 Rev 04 21 NucleoSpin RNA XS Add 200 ul 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 centrifuga tion 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 If higher RNA concentrations or higher elution volumes are desired elution volume may be varied in the range of 5 30 ul For further details on alternative eluti
19. g 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 28 MACHEREY NAGEL 01 2010 Rev 04 Total RNA Isolation Problem Possible cause and suggestions Poor RNA quality or yield continued lonic strength and pH influence A absorption as well as ratio Asl Aog e For adsorption measurement use 5 mM Tris pH 8 5 as diluent Please see also Manchester K L 1995 Value of AJA 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 s
20. hed the NucleoSpin RNA XS Column after centrifuga tion discard flow through and centrifuge again MACHEREY NAGEL 01 2010 Rev 04 17 NucleoSpin RNA XS 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 If higher RNA concentrations or higher elution volumes are desired elution volume may be varied in the range of 5 30 pl For further details on alternative elution procedures see section 2 4 10 pl RNase free H O 11 000 x g 30s 18 MACHEREY NAGEL 01 2010 Rev 04 NucleoSpin RNA XS 5 2 Total RNA purification from tissue with NucleoSpin RNA XS Before starting the preparation e 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 microcentri fuge tube not provided For appropriate sample amounts see section 2 2 2 Lyse and homogenize tissue Add 200 pl Buffer RA1 and 4 jil 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 4 200 ul RA1 4 ul TCEP If multiple samples are processed the preparation of a master premix is recommended e g 2 2 ml Buffer RA1 and 44 ul TCEP for 10
21. m ADP with polynucleotide phosphorylase is included for optimal 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 trans fered into a reverse transcription reaction is commonly not significantly influenc ing the outcome of the reaction due to the 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 If subsequent to total RNA isola tion a poly A RNA isolation is performed adding Carrier RNA should be omitted Other types of carrier RNA may be used in such cases for example bacterial ribosomal RNA 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 Table 1 Kit specifications at a glance Parameter NucleoSpin RNA XS Sample material n ji Typical yield See table 2 for examples Elution volume 5 30 ul Binding capacity 90 ug Maximal loading volume 600 ul Preparation time 45 min 12 preps Format XS spin column MACHEREY NAGEL 01 2010 Rev 04 7 Total RNA Isolation Table 2
22. materials 8 2 4 Elution procedures 10 2 5 Stability of isolated RNA 10 3 Storage conditions and preparation of working solutions 11 4 Safety instructions risk and safety phrases 13 5 Protocols 15 5 1 Total RNA purification from cultured cells laser captured cells or microdissected cryosections with NucleoSpin RNA XS 15 5 2 Total RNA purification from tissue with NucleoSpin RNA XS 19 5 3 Clean up and concentration of RNA with NucleoSpin RNA XS 23 5 4 Support protocol NucleoSpin RNA XS rDNase digestion in the eluate 26 6 Appendix 28 6 1 Troubleshooting 28 6 2 Ordering information 32 6 3 References 33 6 4 Product use restriction warranty 33 MACHEREY NAGEL 01 2010 Rev 04 3 Total RNA Isolation 1 Components 1 1 Kit contents NucleoSpin RNA XS 10 preps 50 preps 250 preps Cat No 740902 10 740902 50 740902 250 Lysis Buffer RA1 2x1 8ml 25 ml 80 ml Wash Buffer RA2 2x1 mi 15 ml 2x15ml Wash Burer RAS 2ml 7 mi 2 x 20 ml Concentrate Membrane Desalting Buffer MDB 1 8 ml 10 ml 50 ml Reaction Buffer for rDNase 0 5 ml 3 ml 20 ml rDNase RNase free 1 vial 1 vial 2 vials lyphilized size A size C size D Carrier RNA 300 ug 300 ug 300 ug Reducing Agent TCEP 14 mg 3x 14 mg 2 x 107 mg RNase free H O 5 ml 15 ml 25 ml NucleoSpin Filters 10 50 250 violet rings NucleoSpin RNA XS Columns light blue rings 10 50 250 plus Collection Tubes Collection Tubes 2 ml 30 150 750 Collection Tubes 1 5
23. ml 10 50 250 User Manual 1 1 1 For preparation of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 01 2010 Rev 04 Total RNA Isolation 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 e 70 ethanol to adjust RNA binding condition Consumables e 1 5 ml microcentrifuge tubes Sterile RNase free tips Equipment Manual pipettors e Centrifuge for microcentrifuge tubes e Vortex mixer e 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 purifica tion procedure All technical literature is available on the internet at www mn net com MACHEREY NAGEL 01 2010 Rev 04 5 Total 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
24. nt 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 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 XS 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 replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business te
25. on 20 21 22 tact with skin and if swallowed MDB Guanidine t Flammable R 10 S 16 thiocyanate lt 10 etha nol lt 10 TCEP Tris 2 car x Xn Causes burns R34 S 26 27 boxylethyl 36 37 39 phosphine Hydrochloride 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 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet Hazard labeling not neccessary if quantity 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 04 13 Total RNA Isolation Risk phrases R 10 R 20 21 22 R 34 R 42 43 Flammable Harmful by inhalation in contact with skin and if swallowed Causes burns May cause sensitisation by inhalation and skin contact Safety phrases 13 S16 S 22 S24 S 26 S27 S 36 37 39 Keep away from food drink and animal feedstuffs Keep away from sources of ignition No Smoking Do not breathe dust Avoid contact with the skin In case of contact with eyes rinse immediately with plenty of water and seek medical advice Take off immediately all contaminated clothing Wear suitable protective clothing glovers and eye face protection 14 MACHEREY N
26. on procedures see section 2 4 200 pl RA3 11 000 x g 30s 10 pl RNase free H O 11 000 x g 30 s 22 MACHEREY NAGEL 01 2010 Rev 04 NucleoSpin RNA XS 5 3 Clean up and concentration of RNA with NucleoSpin RNA XS Before starting the preparation e Check if Wash Buffer RA3 were prepared according to section 3 Supply sample Provide up to 300 pl sample such as prepurified RNA e g phenol purified or RNA from reaction mixtures y i Sample e g labelling reactions in a microcentrifuge tube not provided For appropriate sample amounts see section 2 2 Prepare lysis binding buffer premix 25 pl RAI 75 pl EtOH For every 100 ul of sample combine 25 pl Buffer RA1 96 100 i with 75 pl ethanol 96 100 and mix per 100 pl If processing multiple samples the preparation of a sample master premix 1 volume Buffer RA1 plus 3 volumes ethanol 96 10096 is recommended Mix Add Carrier RNA Not necessary Filtrate lysate Not necessary Adjust RNA binding condition Add 1 vol premix to Add one volume of premix to the sample e g 100 ul sample premix to a 100 ul sample and mix 2 x 5 s If necessary spin down briefly approx 1 s 1000 x g to clear the lid Mix MACHEREY NAGEL 01 2010 Rev 04 23 NucleoSpin RNA XS 6 Bind RNA For each preparation take one NucleoSpin RNA XS Column light blue ring placed in a Coll
27. pectrophotometric assess ment of nucleic acid purity Biotechniques 22 474 481 Sample material e 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 allow tissues to thaw before addition of Buffer RA1 Perform disruption of samples immediately after addition of Lysis Buffer RA1 e Insufficient disruption and or homogenization of starting material Ensure thorough sample disruption and use NucleoSpin Filters for easy homogenization of disrupted starting material Clogged NucleoSpin Column Poor RNA quality or yield Sample material e Too much starting material used Overloading may lead to decreased overall yield Reduce amount of sample material or use larger volume of Buffer RA1 e Insufficient disruption and or homogenization of starting material Ensure thorough sample disruption and use NucleoSpin Filters for easy homogenization of disrupted starting material MACHEREY NAGEL 01 2010 Rev 04 29 Total RNA Isolation Problem Possible cause and suggestions Contamination of RNA with genomic DNA rDNase not active e Reconstitute and store lyophilized rDNase according to instructions given in section 3 DNase solution not properly applied e Pipette rDNase solution directly onto the center of the silica membrane and close the lid Too much cell mat
28. rms 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 MACHEREY NAGEL 01 2010 Rev 04 33 Total RNA Isolation 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 stated specifications This warranty is exclusive and MACHEREY NAGEL mak
29. sible cause and suggestions Suboptimal Store isolated RNA properly performance e Eluted RNA should always be kept on ice for optimal stability of RNA in since trace contaminations of omnipresent RNases general downstream lab ware fingerprints dust will degrade the isolated RNA experiments For short term storage freeze at 20 C for long term storage continued freeze at 70 C Discrepancy between A quantification values and PCR quantifi cation values Silica abrasion from the membrane e Due to the typically low RNA content in very small samples and the resulting low total amount of isolated RNA an RNA quantification via A absorption measurement is often hampered due to the low sensitivity of the absorp tion measurement When performing absorption mea surements close to the detection limit of the photometer the measurement may be influenced by minor amounts of silica abrasion In order to prevent incorrect A quantifica tion of small RNA amounts centrifuge the eluate for 30 s at 211 000 x g and take an aliquot for measurement without disturbing any sediment Alternatively use a silica abrasion insensitive RNA quantification method e g RiboGreen fluo rescent dye Unexpected A A ratio 260 280 Measurement not in the range of photometer detection limit e n order to obtain a significant A A ratio it is necessary that the initially measured A and A values are significantly above th
30. ted 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 finger prints 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 01 2010 Rev 04 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 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 20 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 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 RN
31. ter violet ring in a Collection Tube 2 ml supplied apply the mixture and centrifuge for 30 s at 11 000 x g This step may be skipped when working with small amounts of sample for example less than 105 cells Adjust RNA binding condition Discard the NucleoSpin Filter violet ring Add 100 pl ethanol 70 to the homogenized lysate and mix by pipetting up and down 5 times Alternatively add 100 ul ethanol 70 to the sample in a 1 5 ml microcentrifuge tube not provided 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 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 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 pl 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
32. ws elution of RNA in as little as 5 30 ul Thus highly 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 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 e The NucleoSpin RNA XS kit allows purification of RNA with an A A 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 gener 6 MACHEREY NAGEL 01 2010 Rev 04 Total RNA Isolation ating stronger signal compared to reactions with less template in a LightCycler PCR with the Sigma SYBR Green Quantitative RT PCR Kit 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 fro
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