RNeasy® MinElute® Cleanup Handbook
Contents
1. Buffer RPE is supplied as a concentrate Before using for the first time add 4 volumes of ethanol 96 100 as indicated on the bottle to obtain a working solution Procedure D1 Carry out homogenization of the sample in QIAzol Lysis Reagent followed by phase separation as described in the QIAzol Handbook steps 1 7 of the QIAzol protocol for lysis and homogenization D2 Transfer the upper aqueous phase to a new collection tube Add 1 volume of 70 ethanol and mix thoroughly by vortexing Do not centrifuge Proceed immediately to step D3 D3 Transfer up to 700 pl of the sample to an RNeasy MinElute spin column placed in a 2 ml collection tube supplied Close the lid gently and centrifuge for 15 s at 28000 x g 210 000 rpm Discard the flow through If the sample is gt 700 pl transfer the remaining sample up to 700 pl and repeat the centrifugation Discard the flow through D4 Place the RNeasy MinElute spin column in a new 2 ml collection tube supplied Add 500 pl Buffer RPE to the spin column Close the lid gently and centrifuge for 15 s at gt 8000 x g 210 000 rpm to wash the spin column membrane Discard the flow through Reuse the collection tube in step D5 Note Buffer RPE is supplied as a concentrate Ensure that ethanol is added to Buffer RPE before use see Things to do before starting D5 Add 500 pl of 80 ethanol to the RNeasy MinElute spin column Close the lid gently and centrifuge for 2 min at2. 1500 units RNase Free DNase I 79254 RNase Free Buffer RDD and RNase Free Water Related products RNeasy Plus Micro Kit for purification of total RNA from small cell and tissue samples using gDNA Eliminator columns RNeasy Plus Micro Kit 50 50 RNeasy MinElute Spin Columns 74034 50 gDNA Eliminator Mini Spin Columns Collection Tubes Carrier RNA RNase Free Reagents and Buffers RNeasy Mini Kit for purification of total RNA from cells tissues and yeast and for RNA cleanup RNeasy Mini Kit 50 50 RNeasy Mini Spin Columns 74104 Collection Tubes RNase Free Reagents and Buffers PAXgene Blood RNA Kit for isolation and purification of intracellular RNA from whole blood stabilized in PAXgene Blood RNA Tubes PAXgene Blood RNA Kit 50 50 PAXgene Spin Columns 50 762164 PAXgene Shredder Spin Columns 7621745 Processing Tubes RNase Free DNase Set RNase Free Reagents and Buffers To be used in conjunction with PAXgene Blood RNA Tubes Larger kit size and format available for details visit www giagen com RNA t PAXgene Blood RNA Tubes cat no 762165 are available from BD and BD authorized distributors www bd com Canada and USA 8 Rest of the world kit not available in all countries 28 RNeasy MinElute Cleanup Handbook 10 2010 Ordering Information Product Contents Cat no QlAzol Lysis Reagent for efficient lysis of fatty tissues and all other types of tissue before RNA puri
3. 2x Master Mix contains ROX dye 2 x 2 ml RNase Free Water Larger kit size available for details visit www giagen com PCR RNeasy MinElute Cleanup Handbook 10 2010 29 Ordering Information Product Contents Cat no For the Applied Biosystems 7500 and instruments from Bio Rad MJ Research Cepheid Corbett Research Eppendorf Roche and Stratagene QuantiFast Probe For 400 x 25 pl reactions 3x 1 7 ml 204354 PCR ROX Vial Kit 400 2x Master Mix without ROX dye 210 pl ROX Dye Solution 2 x 2 ml RNase Free Water QuantiFast Probe RT PCR Kits for fast quantitative real time one step RT PCR using sequence specific probes For all instruments from Applied Biosystems except the Applied Biosystems 7500 QuantiFast Probe For 400 x 25 pl reactions 3x 1 7 ml 204454 RT PCR Kit 400 2x Master Mix contains ROX dye 100 pl RT Mix 2 x 2 ml RNase Free Water For the Applied Biosystems 7500 and instruments from Bio Rad MJ Research Cepheid Corbett Research Eppendorf Roche and Stratagene QuantiFast Probe For 400 x 25 pl reactions 3x 1 7 ml 204554 RT PCR ROX Vial Kit 400 2x Master Mix without ROX dye 210 pl ROX Dye Solution 100 yl RT Mix 2 ml RNase Free Water For up to date licensing information and productspecific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or y
4. DNase digestion is not required since the combination of QIAzol and RNeasy MinElute technologies efficiently removes most of the DNA without DNase treatment However further DNA removal may be necessary for certain RNA applications that are sensitive to very small amounts of DNA e g TaqMan RT PCR analysis with a low abundance target If DNase digestion is required we recommend performing RNA cleanup with the RNeasy Micro Kit cat no 74004 which is supplied with the RNase Free DNase Set and Buffer RW1 for on column DNase digestion Please note that the combination of the RNeasy MinElute Cleanup Kit and the RNase Free DNase Set cannot be used for RNA cleanup with on column DNase digestion as they are not supplied with Buffer RW1 HM QlIAzol Lysis Reagent contains a guanidine salt and is therefore not compatible with disinfecting reagents containing bleach See the QIAzol Handbook for safety information This protocol also works well with some other reagents containing phenol and guanidine thiocyanate Please contact QIAGEN Technical Services for more details see back cover for contact information RNeasy MinElute Cleanup Handbook 10 2010 25 M Unless otherwise indicated perform all steps of the procedure at room temperature 15 25 C During the procedure work quickly E Perform all centrifugation steps at 20 25 C in a standard microcentrifuge Ensure that the centrifuge does not cool below 20 C Things to do before starting
5. gt 8000 x g 210 000 rpm to wash the spin column membrane Discard the flow through and collection tube Note After centrifugation carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur Flow through contains QIAzol Lysis Reagent and is therefore not compatible with bleach See the QIAzol Handbook for safety information 26 RNeasy MinElute Cleanup Handbook 10 2010 D D7 Place the RNeasy MinElute spin column in a new 2 ml collection tube supplied Open the lid of the spin column and centrifuge at full speed for 5 min Discard the flow through and collection tube To avoid damage to their lids place the spin columns into the centrifuge with at least one empty position between columns Orient the lids so that they point in a direction opposite to the rotation of the rotor e g if the rotor rotates clockwise orient the lids counterclockwise It is important to dry the spin column membrane since residual ethanol may interfere with downstream reactions Centrifugation with the lids open ensures that no ethanol is carried over during RNA elution Place the RNeasy MinElute spin column in a new 1 5 ml collection tube supplied Add 14 pl RNase free water directly to the center of the spin column membrane Close the lid gently and centrifuge for 1 min at full speed to elute the RNA As litle as 10 pl RNas
6. to wash the spin column membrane Discard the flow through Reuse the collection tube in step 5 Note Buffer RPE is supplied as a concentrate Ensure that ethanol is added to Buffer RPE before use see Things to do before starting 5 Add 500 pl of 80 ethanol to the RNeasy MinElute spin column Close the lid gently and centrifuge for 2 min at gt 8000 x g 210 000 rpm to wash the spin column membrane Discard the flow through and collection tube Note After centrifugation carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur Flow through contains Buffer RLT and is therefore not compatible with bleach See page 6 for safety information RNeasy MinElute Cleanupi Handbook 10 2010 11 a gt e 8 O lt Z Place the RNeasy MinElute spin column in a new 2 ml collection tube supplied Open the lid of the spin column and centrifuge at full speed for 5 min Discard the flow through and collection tube To avoid damage to their lids place the spin columns into the centrifuge with at least one empty position between columns Orient the lids so that they point in a direction opposite to the rotation of the rotor e g if the rotor rotates clockwise orient the lids counterclockwise It is important to dry the spin column membrane since residual ethanol may interfere with downstream reactions
7. free water The reaction volumes can be doubled if necessary to 200 pl final volume C2 Incubate on the benchtop 20 25 C for 10 min C3 Clean up the RNA according to Protocol RNA Cleanup and Concentration on page 10 24 RNeasy MinElute Cleanup Handbook 10 2010 Appendix D RNA Cleanup after Lysis and Homogenization with QIAzol Lysis Reagent QIAzol Lysis Reagent is a monophasic solution of phenol and guanidine thiocyanate that can be used for sample lysis and partial purification of total RNA see page 29 for ordering information Cleanup using the RNeasy MinElute Cleanup Kit is recommended to remove any contaminating phenol The aqueous phase from QIAzol lysis is used as starting material in this protocol It is therefore not necessary to precipitate the RNA and redissolve it prior to RNA cleanup Determining the correct amount of starting material This protocol is designed for QlIAzol preps with a maximum starting volume of 1 ml QIAzol Lysis Reagent This corresponds to a final volume of approximately 600 yl aqueous phase for RNA cleanup A maximum of 45 pg RNA can be cleaned up in this protocol This amount corresponds to the binding capacity of the RNeasy MinElute spin column If the expected RNA yield is gt 45 pg use an appropriate proportion of the QIAzol lysate per RNeasy MinElute spin column Important points before starting E f preparing RNA for the first time read Appendix A page 19 E Generally
8. surface of the skin or from dusty laboratory equipment Change gloves frequently and keep tubes closed whenever possible Keep purified RNA on ice when aliquots are pipetted for downstream applications Disposable plasticware The use of sterile disposable polypropylene tubes is recommended throughout the procedure These tubes are generally RNase free and do not require pretreatment to inactivate RNases Nondisposable plasticware Nondisposable plasticware should be treated before use to ensure that it is RNase free Plasticware should be thoroughly rinsed with 0 1 M NaOH 1 mM EDTA followed by RNase free water see Solutions page 20 Alternatively chloroform resistant plasticware can be rinsed with chloroform to inactivate RNases When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material data sheets MSDSs available from the product suppliers RNeasy MinElute Cleanup Handbook 10 2010 19 Glassware Glassware should be treated before use to ensure that it is RNase free Glassware used for RNA work should be cleaned with a detergent thoroughly rinsed and oven baked at 240 C for at least 4 hours overnight if more convenient before use Autoclaving alone will not fully inactivate many RNases Alternatively glassware can be treated with DEPC diethyl pyrocarbonate Fill glassware with 0 1 DEPC 0 1 in water all
9. to the center of the spin column membrane Close the lid gently and centrifuge for 1 min at full speed to elute the RNA As little as 10 pl elution buffer BR5 can be used for elution if a higher RNA concentration is required but the yield will be reduced by approximately 20 Do not elute with less than 10 pl elution buffer BR5 as the spin column membrane will not be sufficiently hydrated The dead volume of the RNeasy MinElute spin column is 2 pl elution with 14 pl elution buffer BRS results in a 12 pl eluate 9 Incubate the eluate for 5 min at 65 C in a heating block or water bath After incubation chill immediately on ice Co o gt o dnuvp j YNA Denaturation of the eluate is essential for maximum efficiency in downstream applications such as RT PCR other amplification reactions or cDNA synthesis It is not necessary to denature samples more than once and samples remain denatured after freezing and thawing For RT PCR and realtime RT PCR with the purified RNA QIAGEN offers a range of optimized ready to use kits that provide highly specific and sensitive results For details visit www giagen com PCR For whole transcriptome amplification WTA of limited amounts of RNA we recommend the QuantiTect Whole Transcriptome Kit For details visit www giagen com goto WTA RNeasy MinElute Cleanup Handbook 10 2010 15 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems t
10. 2010 3 Kit Contents RNeasy MinElute Cleanup Kit 50 Catalog no 74204 Number of preps 50 RNeasy MinElute Spin Columns each in a 2 ml Collection Tube 50 Collection Tubes 1 5 ml 50 Collection Tubes 2 ml 100 Buffer RLT A5 ml Buffer RPEt concentrate 11 ml RNase Free Water 10 ml Handbook 1 Contains a guanidine salt Not compatible with disinfecting reagents containing bleach See page 6 for safety information t Before using for the first time add 4 volumes of ethanol 96 100 as indicated on the bottle to obtain a working solution Storage Store RNeasy MinElute spin columns immediately upon receipt at 2 8 C Store the remaining components of the RNeasy MinElute Cleanup Kit dry at room temperature 15 25 C All components of the kit are stable for at least 9 months under these conditions Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of RNeasy MinElute Cleanup Kit is tested against predetermined specifications to ensure consistent product quality Product Use Limitations The RNeasy MinElute Cleanup Kit is intended for molecular biology applications This product is not intended for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to oth
11. Centrifugation with the lids open ensures that no ethanol is carried over during RNA elution Place the RNeasy MinElute spin column in a new 1 5 ml collection tube supplied Add 14 pl RNase free water directly to the center of the spin column membrane Close the lid gently and centrifuge for 1 min at full speed to elute the RNA As little as 10 pl RNase free water can be used for elution if a higher RNA concentration is required but the yield will be reduced by approximately 20 Do not elute with less than 10 pl RNase free water as the spin column membrane will not be sufficiently hydrated The dead volume of the RNeasy MinElute spin column is 2 pl elution with 14 pl RNase free water results ina 12 pl eluate For RT PCR and real time RT PCR with the purified RNA QIAGEN offers a range of optimized ready to use kits that provide highly specific and sensitive results For details visit www qiagen com PCR For whole transcriptome amplification WTA of limited amounts of RNA we recommend the QuantiTect Whole Transcriptome Kit For details visit www giagen com goto WTA RNeasy MinElute Cleanup Handbook 10 2010 Protocol Concentration of RNA Purified Using the PAXgene Blood RNA Kit This protocol is for the concentration of total cellular RNA purified using the PAXgene Blood RNA Kit see page 28 for ordering information Determining the correct amount of starting material A maximum of 45 pg RNA in a maximum volume of 200 p
12. Dispense in a fume hood and wear appropriate protective clothing Buffer RLT containing B ME can be stored at room temperature for up to 1 month Alternatively add 20 pl of 2 M dithiothreitol DTT per 1 ml Buffer RLT The stock solution of 2 M DTT in water should be prepared fresh or frozen in single use aliquots Buffer RLT containing DTT can be stored at room temperature for up to 1 month dnup j YNA Procedure 1 Adjust the sample to a volume of A 100 pl or 200 pl with RNase free water Add A 350 pl or 700 pl Buffer RLT and mix well If starting with an RNA pellet be sure that the pellet is dissolved in the RNase free water supplied before adding Buffer RLT Optional Add B ME or DTT to Buffer RLT before use see Things to do before starting 2 Add A 250 pl or 500 pl of 96 100 ethanol to the diluted RNA and mix well by pipetting Do not centrifuge Proceed immediately to step 3 3 Transfer the sample 700 pl to an RNeasy MinElute spin column placed in a 2 ml collection tube supplied Close the lid gently and centrifuge for 15 s at 28000 x g 210 000 rpm Discard the flow through For samples gt 700 pl transfer the remaining sample up to 700 pl and repeat the centrifugation Discard the flow through 4 Place the RNeasy MinElute spin column in a new 2 ml collection tube supplied Add 500 pl Buffer RPE to the spin column Close the lid gently and centrifuge for 15 s at gt 8000 x g 210 000 rpm
13. October 2010 RNeasy MinElute Cleanup Handbook For RNA cleanup and concentration with small elution volumes QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com Contents Kit Contents 4 Storage 4 Quality Control 4 Product Use Limitations 4 Product Warranty and Satisfaction Guarantee 5 Technical Assistance 5 Safety Information 6 Introduction 7 Principle and procedure 8 Automated purification 8 Equipment and Reagents to Be Supplied by User 9 Protocols MRNA Cleanup and Concentration 10 Concentration of RNA Purified Using the PAXgene Blood RNA Kit 13 Troubleshooting Guide 16 Appendix A General Remarks on Handling RNA 19 Appendix B Storage Quantification and Determination of Quality of RNA 21 Appendix C DNase Digestion of RNA before RNA Cleanup 24 Appendix D RNA Cleanup after Lysis and Homogenization with QlAzol Lysis Reagent 25 References 27 Ordering Information 28 RNeasy MinElute Cleanup Handbook 10
14. QIAGEN products If you have any questions or experience any difficulties regarding the RNeasy MinElute Cleanup Kit or QIAGEN products in general please do not hesitate to contact us QIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please see our Technical Support Center at www giagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www giagen com RNeasy MinElute Cleanup Handbook 10 2010 5 Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDSs These are available online in convenient and compact PDF format at www qiagen com Support MSDS aspx where you can find view and print the MSDS for each QIAGEN kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste Buffer RLT contains guanidine thiocyanate which can form highly reactive compounds when combined with bleach If liquid containing this buffer is spilt clean with suitable
15. RNase free pipet tips Microcentrifuge with rotor for 2 ml tubes Ethanol 80 and 96 100 Disposable gloves Optional 14 3 M B mercaptoethanol B ME commercially available solutions are usually 14 3 M or alternatively 2 M dithiothreitol DTT in water For concentration of RNA purified using the PAXgene Blood RNA Kit MH Elution Buffer BR5 included in the PAXgene Blood RNA Kit HM Heating block or water bath capable of reaching 65 C Do not use denatured alcohol which contains other substances such as methanol or methylethylketone RNeasy MinElute Cleanup Handbook 10 2010 9 a gt r S O lt Z Protocol RNA Cleanup and Concentration This protocol is designed for cleaning up RNA from enzymatic reactions for desalting RNA samples and for concentrating RNA isolated by various methods For concentration of total cellular RNA purified using the PAXgene Blood RNA Kit follow the protocol on page 13 Determining the correct amount of starting material A maximum of 45 pg RNA in a maximum volume of 200 pl can be cleaned up in this protocol This amount corresponds to the binding capacity of the RNeasy MinElute spin column Do not overload the column as this will significantly reduce RNA yield and purity Important points before starting E f preparing RNA for the first time read Appendix A page 19 E Generally DNase digestion is not required since RNeasy MinElute silica membrane technology efficien
16. cation Spectrophotometric quantification of RNA To ensure significance Azs readings should be greater than 0 15 An absorbance of 1 unit at 260 nm corresponds to 44 pg of RNA per ml Aggo 1 gt 44 pg ml This relation is valid only for measurements at a neutral pH Therefore if it is necessary to dilute the RNA sample this should be done in a buffer with neutral pH As discussed below see Purity of RNA page 22 the ratio between the absorbance values at 260 and 280 nm gives an estimate of RNA purity When measuring RNA samples be certain that cuvettes are RNase free especially if the RNA is to be recovered after spectrophotometry This can be accomplished by washing cuvettes with 0 1 M NaOH 1 mM EDTA followed by washing with RNase free water see Solutions page 20 Use the buffer in which the RNA is diluted to zero the spectrophotometer An example of the calculation involved in RNA quantification is shown below Volume of RNA sample 10 pl Dilution 1 pl of RNA sample 499 pl of 10 mM Tris Cl pH 7 0 1 500 dilution Measure absorbance of diluted sample in a 1 ml cuvette RNase free A 0 2 Concentration of RNA sample 44 pg ml x Azo x dilution factor 44 pg ml x 0 2 x 500 4400 pg ml When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product sup
17. does not perform well in downstream experiments Ethanol carryover After the wash with 80 ethanol be sure to centrifuge at full speed for 5 min to dry the RNeasy MinElute spin column membrane After centrifugation carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur RNeasy MinElute Cleanup Handbook 10 2010 Appendix A General Remarks on Handling RNA Handling RNA Ribonucleases RNases are very stable and active enzymes that generally do not require cofactors to function Since RNases are difficult to inactivate and even minute amounts are sufficient to destroy RNA do not use any plasticware or glassware without first eliminating possible RNase contamination Great care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure In order to create and maintain an RNase free environment the following precautions must be taken during pretreatment and use of disposable and nondisposable vessels and solutions while working with RNA General handling Proper microbiological aseptic technique should always be used when working with RNA Hands and dust particles may carry bacteria and molds and are the most common sources of RNase contamination Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the
18. e free water can be used for elution if a higher RNA concentration is required but the yield will be reduced by approximately 20 Do not elute with less than 10 pl RNase free water as the spin column membrane will not be sufficiently hydrated The dead volume of the RNeasy MinElute spin column is 2 pl elution with 14 pl RNase free water results in a 12 pl eluate For RT PCR and real time RT PCR with the purified RNA QIAGEN offers a range of optimized ready to use kits that provide highly specific and sensitive results For details visit www giagen com PCR For whole transcriptome amplification WTA of limited amounts of RNA we recommend the QuantiTect Whole Transcriptome Kit For details visit www qiagen com goto WTA References QIAGEN maintains a large up to date online database of scientific publications utilizing QIAGEN products Comprehensive search options allow you to find the articles you need either by a simple keyword search or by specifying the application research area title etc For a complete list of references visit the QIAGEN Reference Database online at www giagen com RefDB search asp or contact QIAGEN Technical Services or your local distributor RNeasy MinElute Cleanup Handbook 10 2010 27 Ordering Information Product Contents Cat no RNeasy MinElute 50 RNeasy MinElute Spin Columns 74204 Cleanup Kit 50 Collection Tubes RNase Free Reagents and Buffers Accessories RNase Free DNase Set 50
19. er applicable guidelines 4 RNeasy MinElute Cleanup Handbook 10 2010 Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its performance and design If a QIAGEN product does not meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover or visit www qiagen com Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of
20. ers 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 800 789 544 Fax 02 334304 826 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 080 000 7146 Fax 02 2626 5703 Technical 080 000 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1 122 330 Technical 01 800 7742 436 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 1800 742 4362 Fax 65 6854 8184 Technical 1800 742 4368 Spain Orders 91 630 7050 Fax 911 630 5145 Technical 91 630 7050 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN ee Sample amp Assay Technologies
21. esidual DEPC must always be eliminated from solutions or vessels by autoclaving or heating to 100 C for 15 minutes Note RNeasy buffers are guaranteed RNase free without using DEPC treatment and are therefore free of any DEPC contamination When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material data sheets MSDSs available from the product suppliers t Plastics used for some electrophoresis tanks are not resistant to ethanol Take proper care and check the supplier s instructions 20 RNeasy MinElute Cleanup Handbook 10 2010 Appendix B Storage Quantification and Determination of Quality of RNA Storage of RNA Purified RNA may be stored at 20 C or 70 C in RNase free water Under these conditions no degradation of RNA is detectable after 1 year Quantification of RNA The concentration of RNA can be determined by measuring the absorbance at 260 nm Asso in a spectrophotometer see Spectrophotometric quantification of RNA below For small amounts of RNA however it may not be possible to accurately determine amounts photometrically Small amounts of RNA can be quantified using an Agilent 2100 bioanalyzer fluorometric quantification or quantitative real time RT PCR When purifying RNA from particularly small samples e g laser microdissected samples quantitative realtime RT PCR should be used for quantifi
22. fication QIAzol Lysis Reagent 200 ml 200 ml QIAzol Lysis Reagent 79306 QuantiTect Reverse Transcription Kit for fast reverse transcription for sensitive real time two step RT PCR QuantiTect Reverse For 50 x 20 pl reactions gDNA 20531 Transcription Kit 50 Wipeout Buffer Quantiscript Reverse Transcriptase Quantiscript RT Buffer RT Primer Mix and RNase Free Water QuantiTect Primer Assays for use in real time RT PCR with SYBR Green detection search for and order assays at www qiagen com GeneGlobe QuantiTect Primer Assay 200 For 200 x 50 pl reactions or Varies 400 x 25 pl reactions 10x QuantiTect Primer Assay lyophilized QuantiFast SYBR Green PCR Kit for fast quantitative real time PCR and two step RT PCR using SYBR Green QuantiFast SYBR Green For 400 x 25 pl reactions 3x 1 7 ml 204054 PCR Kit 400 2x Master Mix contains ROX dye 2 x 2 ml RNase Free Water QuantiFast SYBR Green RT PCR Kit for fast quantitative real time one step RT PCR using SYBR Green QuantiFast SYBR Green For 400 x 25 pl reactions 3x 1 7 ml 204154 RT PCR Kit 400 2x Master Mix contains ROX dye 100 pl RT Mix 2 x 2 ml RNase Free Water QuantiFast Probe PCR Kits for fast quantitative real time PCR and two step RT PCR using sequence specific probes For all instruments from Applied Biosystems except the Applied Biosystems 7500 QuantiFast Probe For 400 x 25 pl reactions 3 x 1 7 ml 204254 PCR Kit 400
23. hat may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www giagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www qiagen com Low or no recovery of RNA a RNase free water or elution buffer BR5 incorrectly dispensed b Ethanol carryover Clogged RNeasy MinElute spin column a Too much starting RNA b Centrifugation temperature too low Comments and suggestions Pipet RNase free water or elution buffer BR5 to the center of the RNeasy MinElute spin column membrane to ensure that the membrane is completely covered After the wash with 80 ethanol be sure to centrifuge at full speed for 5 min to dry the RNeasy MinElute spin column membrane After centrifugation carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur In subsequent preps reduce the amount of starting RNA A maximum of 45 pg RNA can be used including any carrier RNA This amount corresponds to the binding capacity of an RNeasy MinElute spin column The centrifugation temperature should be 20 25 C Some centrifuges may cool to below 20 C even when set at 20 C Thi
24. l RNAs do not sediment efficiently Automated purification Purification of RNA can be fully automated on the QlAcube The innovative QlAcube uses advanced technology to process QIAGEN spin columns enabling seamless integration of automated low throughput sample prep into your laboratory workflow Sample preparation using the QlAcube follows the same steps as the manual procedure i e bind wash and elute enabling you to continue using the RNeasy MinElute Cleanup Kit for cleanup of high quality RNA For more information about the automated procedure see the relevant protocol sheet available at www qiagen com MyQ lAcube The QlAcube is preinstalled with protocols for purification of plasmid DNA genomic DNA RNA viral nucleic acids and proteins plus DNA and RNA cleanup The range of protocols available is continually expanding and additional QIAGEN protocols can be downloaded free of charge at www giagen com MyQlAcube For purification of miRNA and total RNA from a wide range of cells and tissues we recommend using miRNeasy Kits For details visit www qiagen com miRNA 8 RNeasy MinElute Cleanup Handbook 10 2010 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier For all protocols HM Sterile
25. l can be concentrated in this protocol This amount corresponds to the binding capacity of the RNeasy MinElute spin column Before starting the procedure RNA eluates obtained with the PAXgene Blood RNA Kit can be pooled as long as the total amount of RNA is lt 45 pg and the total volume is lt 200 yl Important points before starting The RNA denaturation step 65 C incubation for 5 min of the PAXgene Blood RNA Kit protocol does not need to be performed RNA denaturation takes place in the last step of this protocol Elution buffer BR5 included in the PAXgene Blood RNA Kit is required for RNA elution in this protocol If preparing RNA for the first time read Appendix A page 19 DNase digestion does not need to be performed in this protocol because it has already been carried out in the PAXgene Blood RNA Kit protocol Buffer RLT contains a guanidine salt and is therefore not compatible with disinfecting reagents containing bleach See page 6 for safety information Unless otherwise indicated perform all steps of the procedure at room temperature 15 25 C During the procedure work quickly Perform all centrifugation steps at 20 25 C in a standard microcentrifuge Ensure that the centrifuge does not cool below 20 C In the procedure below A refers to use of starting volumes lt 100 pl and refers to use of starting volumes of 100 200 yl Things to do before starting Buffer RPE is supplied as a concentrate Before using fo
26. laboratory detergent and water If the spilt liquid contains potentially infectious agents clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite The following risk and safety phrases apply to the components of the RNeasy MinElute Cleanup Kit Buffer RLT Contains guanidine thiocyanate harmful Risk and safety phrases R20 21 22 32 13 26 36 46 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 R20 21 22 Harmful by inhalation in contact with skin and if swallowed R32 Contact with acids liberates very toxic gas 13 Keep away from food drink and animal feedingstuffs S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S36 Wear suitable protective clothing S46 If swallowed seek medical advice immediately and show the container or label 6 RNeasy MinElute Cleanup Handbook 10 2010 Introduction The RNeasy MinElute Cleanup Kit uses novel technology to purify and concentrate RNA RNA amounts from 45 pg down to a few picograms which correspond to less than 1 cell can be purified from enzymatic reactions e g in vitro transcription DNase digestion RNA labeling or from RNA samples requiring desalting e g after phenol extraction and ethanol precipitation The kit allows minimal elutio
27. n volumes making it well suited for concentration of RNA e g after RNA purification with the RNeasy Mini Kit or PAXgene Blood RNA Kit QIAGEN provides a wide range of other kits for purification of total RNA from different sample sources for details visit www giagen com RNA RNeasy MinElute Cleanup Procedure Add Buffer RLT and ethanol Bind RNA RNA Wash Elute aut 9 eat Y emt Concentrated RNA solution RNeasy MinElute Cleanup Handbook 10 2010 7 Principle and procedure RNeasy MinElute technology combines the selective binding properties of a silica based membrane with the speed of microspin technology Guanidine thiocyanate containing lysis buffer and ethanol are added to the sample to create conditions that promote selective binding of RNA to the silica membrane of the RNeasy MinElute spin column The sample is then applied to the RNeasy MinElute spin column RNA binds to the silica membrane contaminants are efficiently washed away and high quality RNA is eluted in RNase free water see flowchart With the RNeasy MinElute procedure all RNA molecules longer than 200 nucleotides are purified The procedure provides an enrichment for mRNA since most RNAs lt 200 nucleotides such as 5 85 rRNA 5S rRNA and tRNAs which together comprise 15 20 of total RNA are selectively excluded The size distribution of the purified RNA is comparable to that obtained by centrifugation through a CsCl cushion where smal
28. ngs to do before starting Add 500 pl of 80 ethanol to the RNeasy MinElute spin column Close the lid gently and centrifuge for 2 min at gt 8000 x g 210 000 rpm to wash the spin column membrane Discard the flow through and collection tube Note After centrifugation carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur Place the RNeasy MinElute spin column in a new 2 ml collection tube supplied Open the lid of the spin column and centrifuge at full speed for 5 min Discard the flow through and collection tube To avoid damage to their lids place the spin columns into the centrifuge with at least one emply position between columns Orient the lids so that they point in a direction opposite fo the rotation of the rotor e g if the rotor rotates clockwise orient the lids counterclockwise It is important to dry the spin column membrane since residual ethanol may interfere with downstream reactions Centrifugation with the lids open ensures that no ethanol is carried over during RNA elution Flow through contains Buffer RLT and is therefore not compatible with bleach See page 6 for safety information 14 RNeasy MinElute Cleanup Handbook 10 2010 8 Place the RNeasy MinElute spin column in a new 1 5 ml collection tube supplied Add 14 pl of elution buffer BR5 from the PAXgene Blood RNA Kit directly
29. nol chloroform extraction and alcohol precipitation will contain small amounts of DNA while RNA purified using the PAXgene Blood RNA Kit which includes on column DNase digestion will contain negligible amounts of DNA When analyzing low abundance transcripts by real time RT PCR any interference by residual DNA contamination can be detected by performing a control reaction in which reverse transcriptase is omitted in the RT step To prevent any interference by DNA contamination in real time RT PCR we recommend designing primers that anneal at an exon exon boundary so that genomic DNA will not be amplified QuantiTect Primer Assays from QIAGEN are designed for SYBR Green based real time RT PCR analysis of RNA sequences without detection of genomic DNA where possible the assays can be ordered online at www giagen com GeneGlobe Wilfinger W W Mackey M and Chomezynski P 1997 Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity BioTechniques 22 474 t Values up to 2 3 are routinely obtained for pure RNA in 10 mM Tris Cl pH 7 5 with some spectrophotometers 22 RNeasy MinElute Cleanup Handbook 10 2010 For real time RT PCR assays where amplification of genomic DNA cannot be avoided and for other experiments where RNA must be free of genomic DNA contamination we recommend treating RNA samples with DNase If handling RNA redissolved in water or buffer we recommend performing a DNa
30. nup Handbook 10 2010 23 Appendix C DNase Digestion of RNA before RNA Cleanup This protocol describes how to digest contaminating DNA in RNA solutions prior to RNA cleanup and concentration This protocol requires use of the QIAGEN RNase free DNase Set see page 28 for ordering information see the RNase Free DNase Set product insert for product description and more information Important points before starting E Do not vortex reconstituted DNase I DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the vial Things to do before starting E Prepare DNase stock solution before using the RNase Free DNase Set for the first time Dissolve the lyophilized DNase 1500 Kunitz units in 550 pl of the RNase free water provided To avoid loss of DNase do not open the vial Inject RNase free water into the vial using an RNase free needle and syringe Mix gently by inverting the vial Do not vortex E For long term storage of reconstituted DNase remove the stock solution from the glass vial divide it into single use aliquots and store at 20 C for up to 9 months Thawed aliquots can be stored at 2 8 C for up to 6 weeks Do not refreeze the aliquots after thawing Procedure C1 Mix the following in a microcentrifuge tube m lt 87 5 pl RNA solution contaminated with genomic DNA m 10 pl Buffer RDD m 2 5 pl DNase I stock solution Make the volume up to 100 pl with RNase
31. our local distributor Visit www giagen com geneXpression to find out more about standardized solutions for gene expression analysis from RNA preparation to real time RT PCR Larger kit size available for details visit www giagen com PCR 30 RNeasy MinElute Cleanup Handbook 10 2010 Trademarks QIAGEN QlAcube QIAzol MinElute QuantiFast Quantiscript QuantiTect RNeasy QIAGEN Group Agilent Agilent Technologies Inc Applied Biosystems Applera Corporation or its subsidiaries PAXgene PreAnalytiX GmbH SYBR Molecular Probes Inc TaqMan Roche Group QlAzol Lysis Reagent is a subject of US Patent No 5 346 994 and foreign equivalents Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the RNeasy MinElute Cleanup Kit to the following terms 1 The RNeasy MinElute Cleanup Kit may be used solely in accordance with the RNeasy MinElute Cleanup Handbook and for use with components contained in the Kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the RNeasy MinElute Cleanup Handbook and additional protocols available at www giagen com Other than expressly stated licenses QIAGEN makes no warranty that this Kit and or its use s do not infringe the rights of third parties This Kit and its component
32. ow to stand overnight 12 hours at 37 C and then autoclave or heat to 100 C for 15 minutes to eliminate residual DEPC Electrophoresis tanks Electrophoresis tanks should be cleaned with detergent solution e g 0 5 SDS thoroughly rinsed with RNase free water and then rinsed with ethanol and allowed to dry Solutions Solutions water and other solutions should be treated with 0 1 DEPC DEPC is a strong but not absolute inhibitor of RNases It is commonly used at a concentration of 0 1 to inactivate RNases on glass or plasticware or to create RNase free solutions and water DEPC inactivates RNases by covalent modification Add 0 1 ml DEPC to 100 ml of the solution to be treated and shake vigorously to bring the DEPC into solution Let the solution incubate for 12 hours at 37 C Autoclave for 15 minutes to remove any trace of DEPC DEPC will react with primary amines and cannot be used directly to treat Tris buffers DEPC is highly unstable in the presence of Tris buffers and decomposes rapidly into ethanol and CO When preparing Tris buffers treat water with DEPC first and then dissolve Tris to make the appropriate buffer Trace amounts of DEPC will modify purine residues in RNA by carbethoxylation Carbethoxylated RNA is translated with very low efficiency in cell free systems However its ability to form DNA RNA or RNA RNA hybrids is not seriously affected unless a large fraction of the purine residues have been modified R
33. plier RNeasy MinElute Cleanup Handbook 10 2010 21 Total amount concentration x volume in milliliters 4400 pg ml x 0 01 ml 44 pg of RNA Purity of RNA The ratio of the readings at 260 nm and 280 nm 4360 4250 provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV spectrum such as protein However the Azgo Azgo ratio is influenced considerably by pH Since water is not buffered the pH and the resulting Aggo Azgo ratio can vary greatly Lower pH results in a lower Apgo Azgo ratio and reduced sensitivity to protein contamination For accurate values we recommend measuring absorbance in 10 mM Tris Cl pH 7 5 Pure RNA has an Aj6o Azgo ratio of 1 9 2 1t in 10 mM Tris Cl pH 7 5 Always be sure to calibrate the spectrophotometer with the same solution used for dilution For determination of RNA concentration however we recommend dilution of the sample in a buffer with neutral pH since the relationship between absorbance and concentration Azs reading of 1 44 pg ml RNA is based on an extinction coefficient calculated for RNA at neutral pH see Spectrophotometric quantification of RNA page 21 DNA contamination No currently available purification method can guarantee that RNA is completely free of DNA even when it is not visible on an agarose gel The level of genomic DNA contamination in an RNA sample depends on the method used for RNA purification For example RNA purified by phe
34. r the first time add 4 volumes of ethanol 96 100 as indicated on the bottle to obtain a working solution Buffer RLT may form a precipitate during storage If necessary redissolve by warming and then place at room temperature 15 25 C RNeasy MinElute Cleanup Handbook 10 2010 13 5 Co i Ss dnupa gt VNU S Se 2D aa lt zo Z Procedure Heat a heating block or water bath to 65 C for use in step 9 Adjust the sample to a volume of A 100 pl or 200 pl with RNase free water Add A 350 pl or 700 pl Buffer RLT and mix well Add A 250 pl or 500 pl of 96 100 ethanol to the diluted RNA and mix well by pipetting Do not centrifuge Proceed immediately to step 4 Transfer the sample 700 pl to an RNeasy MinElute spin column placed in a 2 ml collection tube supplied Close the lid gently and centrifuge for 15 s at 28000 x g 210 000 rpm Discard the flow through For samples gt 700 ul transfer the remaining sample up to 700 pl and repeat the centrifugation Discard the flow through Place the RNeasy MinElute spin column in a new 2 ml collection tube supplied Add 500 pl Buffer RPE to the spin column Close the lid gently and centrifuge for 15 s at gt 8000 x g 210 000 rpm to wash the spin column membrane Discard the flow through Reuse the collection tube in step 6 Note Buffer RPE is supplied as a concentrate Ensure that ethanol is added to Buffer RPE before use see Thi
35. s can cause formation of precipitates that can clog the spin column If this happens set the centrifugation temperature to 25 C RNeasy MinElute Cleanup Handbook 10 2010 Comments and suggestions Low Apgo Aogo Value Water used to dilute RNA for Use 10 mM Tris Cl pH 7 5 not RNase free A20 Argo Measurement water to dilute the sample before measuring purity see Appendix B page 21 RNA degraded RNase contamination Although all RNeasy buffers have been tested and are guaranteed RNase free RNases can be introduced during use Be certain not to introduce any RNases during RNA cleanup or later handling See Appendix A page 19 for general remarks on handling RNA Do not put RNA samples into a vacuum dryer or microcentrifuge that has been used in DNA preparations where RNases may have been used Adding B ME to Buffer RLT may be helpful when cleaning up crude preps of RNA e g after salting out methods or samples that contain large amounts of RNases see protocols DNA contamination in downstream experiments No DNase treatment DNase digest the RNA sample before RNA cleanup see Appendix C page 24 For real time two step RT PCR experiments carry out the RT step using the QuantiTect Reverse Transcription Kit which provides cDNA synthesis with integrated removal of genomic DNA contamination For ordering information see page 29 RNeasy MinElute Cleanup Handbook 10 2010 17 Comments and suggestions RNA
36. s are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated aN The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and or its components For updated license terms see www qiagen com 2003 2010 QIAGEN all rights reserved www qiagen com Australia Orders 1 800 243 800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 86 21 3865 3865 Fax 86 21 3865 3965 Technical 800 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany Ord
37. se digestion in solution see Appendix C page 24 before starting the RNA cleanup procedure on page 10 However if the RNA was isolated by phenol chloroform extraction the aqueous phase containing RNA should be subjected to RNA cleanup with on column DNase digestion using the RNeasy Micro Kit cat no 74004 As an alternative to DNase digestion the QuantiTect Reverse Transcription Kit provides fast cDNA synthesis with integrated removal of genomic DNA contamination see ordering information page 29 The kit is dedicated for use in real time two step RT PCR providing high yields of cDNA representing all transcript regions Integrity of RNA The integrity and size distribution of total RNA purified with RNeasy Kits can be checked by denaturing agarose gel electrophoresis and ethidium bromide staining or by using an Agilent 2100 bioanalyzer The respective ribosomal RNAs should appear as sharp bands or peaks The apparent ratio of 28S rRNA to 18S rRNA should be approximately 2 1 If the ribosomal bands or peaks of a specific sample are not sharp but appear as a smear towards smaller sized RNAs it is likely that the RNA sample suffered major degradation either before or during RNA purification When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier RNeasy MinElute Clea
38. tly removes most of the DNA without DNase treatment However further DNA removal may be necessary for certain RNA applications that are sensitive to very small amounts of DNA e g TaqMan RT PCR analysis with a low abundance target In these cases DNA can be removed by a DNase digestion before starting RNA cleanup see Appendix C page 24 M Buffer RLT contains a guanidine salt and is therefore not compatible with disinfecting reagents containing bleach See page 6 for safety information E Perform all steps of the procedure at room temperature 15 25 C During the procedure work quickly E Perform all centrifugation steps at 20 25 C in a standard microcentrifuge Ensure that the centrifuge does not cool below 20 C MH In the procedure below A refers to use of starting volumes lt 100 pl and refers to use of starting volumes of 100 200 pl Things to do before starting M Buffer RPE is supplied as a concentrate Before using for the first time add 4 volumes of ethanol 96 100 as indicated on the bottle to obtain a working solution E Buffer RLT may form a precipitate during storage If necessary redissolve by warming and then place at room temperature 15 25 C 10 RNeasy MinElute Cleanup Handbook 10 2010 E Optional If cleaning up crude RNA preps e g after salting out methods or samples rich in RNases we recommend adding B mercaptoethanol B ME to Buffer RLT before use Add 10 pl B ME per 1 ml Buffer RLT
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