Sample & Assay Technologies RNeasy® Plus Universal
Contents
1. Low A260 A280 value a Not enough QIAzol Lysis Reagent used for homogenization 28 Add RNase free water to the center of the RNeasy spin column membrane to ensure that the membrane is completely covered In subsequent preparations reduce the amount of starting material and or increase the volume of QIAzol Lysis Reagent and the homogenization time RNeasy Plus Universal Handbook 09 2010 Comments and suggestions b Sample not incubated Place the sample at room temperature for 5 min after 15 25 C for 5 min after homogenization as homogenization indicated in the protocol step 4 This step is important to promote dissociation of nucleoprotein complexes c Water used to dilute Use 10 mM Tris Cl pH 7 5 not RNase free RNA for A o A a0o water to dilute the sample before measuring measurement purity see Appendix B page 33 RNA degraded a Inappropriate handling For frozen tissue samples ensure that they were of starting material flash frozen immediately in liquid nitrogen and properly stored at 70 C Perform the RNeasy procedure quickly especially the first few steps See Appendix A page 31 and Handling and storing starting material page 13 b 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 the RNeasy procedure or later handling See Appendix A page 31 for ge2. QuantiTect Reverse Transcription Kit which provides cDNA synthesis with integrated removal of genomic DNA contamination see ordering information page 42 M QlAzol Lysis Reagent and Buffer RWT contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach See page 6 for safety information E Except for phase separation step 8 all protocol and centrifugation steps should be performed at room temperature 15 25 C During the procedure work quickly Things to do before starting E Buffer RWT is supplied as a concentrate Before using for the first time add 2 volumes of ethanol 96 100 as indicated on the bottle to obtain a working solution Buffer RPE is supplied as a concentrate Before using for the first time add 4 volumes of ethanol 96 10096 as indicated on the bottle to obtain a working solution Procedure 1 If using a TissueLyser system add one stainless steel bead 5 mm mean diameter per 2 ml microcentrifuge tube not supplied If working with tissues that are not stabilized in RNAlater or Allprotect Reagent place the tubes on dry ice Note When disrupting tough or very tough samples with the TissueLyser LT we recommend using one or two 7 mm stainless steel beads 2 Excise the tissue sample from the animal or remove it from storage Determine the amount of tissue Do not use more than 50 mg tissue or more than 100 mg brain or adipose tissue Proceed imme
3. 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 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 Plus Universal Handbook 09 2010 31 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 as described in Solutions below 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 0 196 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
4. for North America and Japan t 235 V 50 60 Hz for Europe excluding UK and Ireland 235 V 50 60 Hz for UK and Ireland 235 V 50 60 Hz for Australia SSE L LLLLALLLLLLLLLLLLLLLLLOILLLOOLLLZLCLZZZZLLCENI RNeasy Plus Universal Handbook 09 2010 41 Product Contents Cat no Stainless Steel Beads Stainless Steel Beads suitable for use 69989 5 mm 200 with TissueLyser systems Stainless Steel Beads Stainless Steel Beads suitable for use 69990 7 mm 200 with TissueLyser systems RNase Free DNase Set For 50 RNA minipreps 1500 units 79254 50 RNase Free DNase RNase Free Buffer RDD and RNase Free Water Collection Tubes 2 ml 1000 x 2 ml Collection Tubes 19201 QuantiTect Reverse Trial kit for 10 x 20 ul reactions gDNA 205310 Transcription Kit 10 Wipeout Buffer Quantiscript Reverse Transcriptase Quantiscript RT Buffer RT Primer Mix and RNase Free Water QuantiTect Reverse For 50 x 20 ul reactions gDNA 205311 Transcription Kit 50 Wipeout Buffer Quantiscript Reverse Transcriptase Quantiscript RT Buffer RT Primer Mix and RNase Free Water QuantiTect Reverse For 200 x 20 ul reactions gDNA 205313 Transcription Kit 200 Wipeout Buffer Quantiscript Reverse Transcriptase Quantiscript RT Buffer RT Primer Mix and RNase Free Water For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are avai
5. 79306 74881 RNeasy Plus Universal Handbook 09 2010 Product Contents Cat no TissueRuptor Handheld rotor stator homogenizer 9001271 5 TissueRuptor Disposable Probes 9001272t 9001273 90012748 TissueRuptor 25 nonsterile plastic disposable probes 990890 Disposable Probes 25 for use with the TissueRuptor TissueLyser LT Compact bead mill 100 240 V AC 85600 50 60 Hz requires the TissueLyser LT Adapter 12 Tube available separately TissueLyser LT Adapter Adapter for disruption of up to 12 69980 12 Tube samples in 2 ml microcentrifuge tubes on the Tissuelyser LT Sample Tubes RB 2 ml 1000 safe lock microcentrifuge tubes 990381 2 ml for use with the TissueLyser LT TissueLyser II Bead mill 100 120 220 240 V 85300 50 60 Hz requires the TissueLyser Adapter Set 2 x 24 or TissueLyser Adapter Set 2 x 96 available separately TissueLyser Adapter Set 2 sets of Adapter Plates and 2 racks for 69982 2x 24 use with 2 ml microcentrifuge tubes on the TissueLyser Il TissueLyser Adapter Set 2 sets of Adapter Plates for use with 69984 2x96 Collection Microtubes racked on the TissueLyser II Collection Microtubes Nonsterile polypropylene tubes 1 2 19560 racked ml 960 in racks of 96 Collection Microtube Nonsterile polypropylene caps for 19566 Caps 120 x 8 collection microtubes 1 2 ml 960 in strips of 8 TissueLyser Single Bead For dispensing individual beads 5 mm 69965 Dispenser 5 mm diameter 120 V 60 Hz
6. 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 1122 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 91 630 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 e Q e e UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 RGG e USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN T Sample amp Assay Technologies
7. Plus Universal Kits should be stored dry at room temperature 15 25 C All components are stable for at least 9 months under these conditions QIAzol Lysis Reagent can be stored at room temperature or at 2 8 C Product Use Limitations RNeasy Plus Universal Kits are intended for molecular biology applications These products are not intended for the diagnosis treatment or prevention 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 other applicable guidelines 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 inf
8. RNA wait for 1 min and then centrifuge for 3 min at 3000 5000 x g Flow through contains QIAzol Lysis Reagent or Buffer RWT and is therefore not compatible with bleach See page 6 for safety information RNeasy Plus Universal Handbook 09 2010 25 Table 3 Volumes of RNase free water for RNA elution Expected total RNA yield RNase free water x150 ug 150 ul 150 ug 1 mg 250 ul 17 Repeat step 16 using another volume of RNase free water or using the eluate from step 16 if high RNA concentration is required Reuse the collection tube from step 16 If using the eluate from step 16 the RNA yield will be 15 30 less than that obtained using a second volume of RNase free water but the final RNA concentration will be higher EEREEEEERIUEEIU AL LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLALALLLLLLELLLL UL L ZLLLLCLLLZLZLLLCCLLALLZLLLZLLZZLZLZLZLLZZLZZLLCL A ELCLAZZLZZAM 26 RNeasy Plus Universal Handbook 09 2010 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www qgiagen 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 giagen com Comments and suggestio
9. combination with the TissueLyser Adapter Set 2 x 96 which holds 192 x 1 2 ml microtubes containing stainless steel beads of 5 mm mean diameter In this case we recommend using the RNeasy 96 Universal Tissue Kit which provides high throughput RNA purification from all types of tissue in 96 well format For ordering information see page 40 RNeasy Plus Universal Handbook 09 2010 15 Protocol Purification of Total RNA Using the RNeasy Plus Universal Mini Kit Determining the correct amount of starting material It is essential to use the correct amount of tissue in order to obtain optimal RNA yield and purity With the RNeasy Plus Universal Mini Kit a maximum of 50 mg tissue can generally be processed Using this amount the RNA binding capacity of the RNeasy Mini spin column and the lysing capacity of QlAzol Lysis Reagent will not be exceeded For brain or adipose tissue a maximum of 100 mg tissue can generally be used For tissues with high RNA content such as liver spleen and thymus we recommend using no more than 30 mg tissue to ensure optimal RNA yields and to avoid exceeding the binding capacity of the RNA spin column Average RNA yields from various tissues are given in Table 2 page 13 If there is no information about the nature of your starting material we recommend starting with no more than 30 mg tissue Depending on RNA yield and purity it may be possible to use up to 100 mg tissue in subsequent preparations Do not over
10. the RNeasy spin column Close the lid gently and centrifuge for 2 min at 28000 x g 210 000 rpm to wash the membrane The long centrifugation dries the spin column membrane ensuring that no ethanol is carried over during RNA elution Residual ethanol may interfere with downstream reactions Note After centrifugation carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur 16 Optional Place the RNeasy spin column in a new 2 ml collection tube supplied and discard the old collection tube with the flow through Close the lid gently and centrifuge at full speed for 1 min Perform this step to eliminate any possible carryover of Buffer RPE or if residual flow through remains on the outside of the RNeasy spin column after step 15 17 Place the RNeasy spin column in a new 1 5 ml collection tube supplied Add 30 50 ul RNase free water directly to the spin column membrane Close the lid gently To elute the RNA centrifuge for 1 min at 28000 x g 210 000 rpm 18 Repeat step 17 using another volume of RNase free water or using the eluate from step 17 if high RNA concentration is required Reuse the collection tube from step 17 If using the eluate from step 17 the RNA yield will be 15 30 less than that obtained using a second volume of RNase free water but the final RNA concentration will be higher RNeasy Plus Univers
11. 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 Residual 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 safety data sheets MSDSs available from the product supplier 32 RNeasy Plus Universal Handbook 09 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 should be determined by measuring the absorbance at 260 nm A260 in a spectrophotometer see Spectrophot
12. Generally round bottomed tubes allow more efficient disruption and homogenization than conical bottomed tubes 3 Place the tip of the TissueRuptor disposable probe into the vessel and operate the TissueRuptor at full speed until the lysate is uniformly homogeneous usually 30 60 s RNeasy Plus Universal Handbook 09 2010 23 Note To avoid damage to the TissueRuptor and disposable probe during operation make sure the tip of the probe remains submerged in the buffer Note Incomplete homogenization leads to significantly reduced RNA yields and can cause clogging of the RNeasy spin column Foaming may occur during homogenization especially of brain tissue If this occurs let the homogenate stand at room temperature for 2 3 min until the foam subsides before continuing with the procedure 4 Place the tube containing the homogenate on the benchtop at room temperature 15 25 C for 5 min This step promotes dissociation of nucleoprotein complexes 5 Add 500 pl gDNA Eliminator Solution Securely cap the tube containing the homogenate and shake it vigorously for 15 s Addition of gDNA Eliminator Solution will effectively reduce genomic DNA contamination of the aqueous phase making further treatment with DNase unnecessary 6 Add 1 ml chloroform Securely cap the tube containing the homogenate and shake it vigorously for 15 s Thorough mixing is important for subsequent phase separation 7 Place the tube containing the homog
13. Kit see Appendix C page 36 Information about purification of total RNA containing small RNAs such as miRNA using the RNeasy Plus Universal Midi Kit is provided in Appendix D page 38 QIAGEN also provides miRNeasy Kits for purification of miRNA either in a total RNA fraction or in a fraction enriched in small RNAs miRNeasy Kits are available in both spin column and 96 well formats For more details visit www qiagen com miRNA or ELLLLLIL LELELLLLLELLLLZLLLLLLLILLLULULILLILIILLU ILLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLALAAALTLLLZZZLZZZZZLCENI RNeasy Plus Universal Handbook 09 2010 9 RNeasy Plus Universal RNeasy Plus Universal Mini Procedure Midi Procedure Tissue Tissue l Y w Lyse and P ze l Lyse and homogenize Add gDNA Eliminator Solution Y P and chloroform w and shake Add gDNA e Separate phases ueri nl i and shake 1 em Separate phases Add ethanol to l aqueous phase ww Bind total RNA Add ethanol to e y aqueous phase w Wash Bind total RNA e Elute Total RNA Wash Elute Total RNA 10 RNeasy Plus Universal Handbook 09 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 SW Chloroform Ethanol 70 and 96 10096 Sterile RNase free pipet tips For stabil
14. September 2010 RNeasy Plus Universal Handbook RNeasy Plus Universal Mini Kit RNeasy Plus Universal Midi Kit For purification of total RNA from all types of tissue QIAGEN Sample amp Assay Technologies 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 5 Product Use Limitations 5 Product Warranty and Satisfaction Guarantee 5 Technical Assistance 5 Quality Control 6 Safety Information 6 Introduction 8 Principle and procedure 8 Equipment and Reagents to Be Supplied by User 11 Important Notes 12 Determining the amount of starting material 12 Handling and storing starting material 13 Disrupting and homogenizing starting material 14 Protocols E Purification of Total RNA Using the RNeasy Plus Universal Mini Kit 16 E Purification of Total RNA Using the RNeasy Plus Universal Midi Kit 22 Troubleshooting Guide 27 Appendix A General Remarks on Handling RNA 31 A
15. al Handbook 09 2010 21 Protocol Purification of Total RNA Using the RNeasy Plus Universal Midi Kit Determining the correct amount of starting material It is essential to use the correct amount of tissue in order to obtain optimal RNA yield and purity With the RNeasy Plus Universal Midi Kit a maximum of 250 mg tissue can generally be processed Using this amount the RNA binding capacity of the RNeasy Midi spin column and the lysing capacity of QIAzol Lysis Reagent will not be exceeded For brain or adipose tissue a maximum of 500 mg tissue can generally be used For tissues with high RNA content such as liver spleen and thymus we recommend using no more than 150 mg tissue to ensure optimal RNA yields and to avoid exceeding the binding capacity of the RNA spin column Average RNA yields from various tissues are given in Table 2 page 13 If there is no information about the nature of your starting material we recommend starting with no more than 150 mg tissue Depending on RNA yield and purity it may be possible to use up to 500 mg tissue in subsequent preparations Do not overload the RNeasy spin column as this will significantly reduce RNA yield and quality Weighing tissue is the most accurate way to quantify the amount of starting material As a guide a 6 mm cube 216 mm of most animal tissues weighs 240 280 mg Important points before starting M If using RNeasy Plus Universal Kits for the first time read Important N
16. and homogenized in the presence of RNase inhibiting or denaturing reagents Otherwise unwanted changes in the gene expression profile will occur It is therefore important that tissue samples are immediately frozen in liquid nitrogen and stored at 70 C or immediately immersed in RNAlater RNA Stabilization Reagent at room temperature 15 25 C An alternative to RNAlater RNA Stabilization Reagent is Allprotect Tissue Reagent which provides immediate stabilization of DNA RNA and protein in tissue samples at room temperature Note RNAlater RNA Stabilization Reagent cannot be used to stabilize RNA in adipose tissue due to the high abundance of fat but can be used to stabilize RNA in other fatty tissues such as brain Allprotect Tissue Reagent can stabilize adipose and brain tissue The procedures for tissue harvesting and RNA protection should be carried out as quickly as possible Frozen tissue samples should not be allowed to thaw during handling or weighing e RNeasy Plus Universal Handbook 09 2010 13 Disrupting and homogenizing starting material Efficient disruption and homogenization of the starting material is an absolute requirement for all total RNA purification procedures Disruption and homogenization are 2 distinct steps M Disruption Complete disruption of plasma membranes of cells and organelles is absolutely required to release all the RNA contained in the sample Incomplete disruption results in significantly reduc
17. ash the membrane The long centrifugation dries the spin column membrane ensuring that no ethanol is carried over during RNA elution Residual ethanol may interfere with downstream reactions Note After centrifugation carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur D7 Place the RNeasy spin column in a new 15 ml collection tube supplied Add the appropriate volume of RNase free water see Table 4 directly to the spin column membrane Close the lid gently To elute the RNA wait for 1 min and then centrifuge for 3 min at 3000 5000 x g Table 4 Volumes of RNase free water for RNA elution Expected total RNA yield RNase free water lt 150 ug 150 ul 150 ug 1 mg 250 ul D8 Repeat step D7 using another volume of RNase free water or using the eluate from step D7 if high RNA concentration is required Reuse the collection tube from step D7 If using the eluate from step D7 the RNA yield will be 15 30 less than that obtained using a second volume of RNase free water but the final RNA concentration will be higher or ELELLIL LELEZLLELLLELLLLZLLLLILLLLLLLILLILIILILUI LLLUALLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLALLLLLLLLLLLLLALALALLLLZZZZZZLCCUXI RNeasy Plus Universal Handbook 09 2010 39 Ordering Information Product RNeasy Plus Universal Mini Kit 50 RNeasy Plus Universal Midi Kit 10 Related p
18. d and can be extended until the tissue is completely homogenized E Carefully pipet the lysates into new microcentrifuge tubes not supplied Proceed to step 4 Do not reuse the stainless steel beads 3c Disruption and homogenization using the TissueLyser II E Place the tissues in the tubes prepared in step 1 E If the tubes were stored on dry ice place them at room temperature Then immediately add 900 pl QIAzol Lysis Reagent per tube E Place the tubes in the TissueLyser Adapter Set 2 x 24 E Operate the TissueLyser for 2 min at 20 Hz The time depends on the tissue being processed and can be extended until the tissue is completely homogenized E Disassemble the adapter set rotate the rack of tubes so that the tubes nearest to the TissueLyser II are now outermost and reassemble the adapter set Operate the TissueLyser for another 2 min at 20 Hz Rearranging the tubes allows even homogenization E Carefully pipet the lysates into new microcentrifuge tubes not supplied Proceed to step 4 Do not reuse the stainless steel beads 4 Place the tube containing the homogenate on the benchtop at room temperature 15 25 C for 5 min This step promotes dissociation of nucleoprotein complexes 5 Add 100 pl gDNA Eliminator Solution Securely cap the tube containing the homogenate and shake it vigorously for 15 s Addition of gDNA Eliminator Solution will effectively reduce genomic DNA contamination of the aqueous phase making f
19. diately to step 3 Weighing tissue is the most accurate way to determine the amount If the tissue sample was stored in RNAlater or Allprotect Reagent remove it from the reagent using forceps and be sure to remove any excess reagent or crystals that may have formed RNA in harvested tissues is not protected until the tissues are treated with RNAlater or Allprotect Reagent flash frozen or disrupted and homogenized in step 3 Frozen tissues should not be allowed to thaw during handling The relevant procedures should be carried out as quickly as possible I RNeasy Plus Universal Handbook 09 2010 17 3 Disrupt the tissue and homogenize the lysate using the TissueRupter follow step 3a TissueLyser LT follow step 3b or TissueLyser II follow step 3c See Disrupting and homogenizing starting material page 14 for more details on disruption and homogenization Note Incomplete homogenization leads to significantly reduced RNA yields and can cause clogging of the RNeasy spin column Homogenization with TissueRupter and TissueLyser systems generally results in higher RNA yields than with other methods 3a Disruption and homogenization using the TissueRuptor E Place the tissue in a suitably sized vessel containing 900 pl QIAzol Lysis Reagent Note Use a suitably sized vessel with sufficient extra headspace to accommodate foaming which may occur during homogenization Generally round bottomed tubes allow more efficient disrupt
20. ed RNA yields E Homogenization Homogenization is necessary to reduce the viscosity of the lysates produced by disruption Homogenization shears high molecular weight genomic DNA and other high molecular weight cellular components to create a homogeneous lysate Incomplete homogenization results in inefficient binding of RNA to the RNeasy spin column membrane and therefore significantly reduced RNA yields Disruption and homogenization of tissue samples can be carried out rapidly and efficiently using either the TissueRuptor for processing samples individually or a TissueLyser system for processing multiple samples simultaneously Disruption and homogenization with TissueRuptor and TissueLyser systems generally results in higher RNA yields than with other methods Disruption and homogenization using the TissueRuptor The TissueRuptor is a rotor stator homogenizer that thoroughly disrupts and simultaneously homogenizes single tissue samples in the presence of lysis buffer in 15 90 seconds depending on the toughness and size of the sample The blade of the TissueRuptor disposable probe rotates at a very high speed causing the sample to be disrupted and homogenized by a combination of turbulence and mechanical shearing For guidelines on using the TissueRuptor refer to the TissueRuptor Handbook For other rotor stator homogenizers refer to suppliers guidelines Disruption and homogenization using TissueLyser systems In bead milling ti
21. ed in a 2 ml collection tube supplied Close the lid gently and centrifuge for 15 s at 28000 x g 210 000 rpm at room temperature 15 25 C Discard the flow through Reuse the collection tube in step 12 12 Repeat step 11 using the remainder of the sample Discard the flow through Reuse the collection tube in step 13 13 Add 700 pl Buffer RWT to the RNeasy spin column Close the lid gently and centrifuge for 15 s at 28000 x g 210 000 rpm to wash the membrane Discard the flow through Reuse the collection tube in step 14 After centrifugation carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow through Be sure to empty the collection tube completely Note Buffer RWT is supplied as a concentrate Ensure that ethanol is added to Buffer RWT before use see Things to do before starting page 17 Flow through contains QIAzol Lysis Reagent or Buffer RWT and is therefore not compatible with bleach See page 6 for safety information 20 RNeasy Plus Universal Handbook 09 2010 14 Add 500 pl Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge for 15 s at 28000 x g 210 000 rpm to wash the membrane Discard the flow through Reuse the collection tube in step 15 Note Buffer RPE is supplied as a concentrate Ensure that ethanol is added to Buffer RPE before use see Things to do before starting page 17 15 Add 500 pl Buffer RPE to
22. enate on the benchtop at room temperature for 2 3 min 8 Centrifuge at 5000 x g for 15 min at 4 C After centrifugation heat the centrifuge to room temperature 15 25 C if the same centrifuge will be used in the later steps of this procedure After centrifugation the sample separates into 3 phases an upper colorless aqueous phase containing RNA a white interphase and a lower red organic phase For tissues with an especially high fat content an additional clear phase may be visible below the red organic phase The volume of the aqueous phase should be approximately 3 ml 9 Transfer the upper aqueous phase to a new tube not supplied 10 Add 1 volume usually 3 ml of 7096 ethanol and mix thoroughly by vortexing Do not centrifuge Proceed immediately to step 11 Note The volume of lysate may be less than 3 ml due to loss during homogenization and centrifugation Precipitates may be visible after addition of ethanol Resuspend precipitates completely by vigorous shaking and proceed immediately to step 11 24 RNeasy Plus Universal Handbook 09 2010 11 Transfer up to 4 ml of the sample to an RNeasy Midi spin column placed in a 15 ml collection tube supplied Close the lid gently and centrifuge for 5 min at 3000 5000 x g at room temperature 15 25 C Discard the flow through Reuse the collection tube in step 12 12 Repeat step 11 using the remainder of the sample up to 4 ml Discard the flow through Reuse t
23. er than those expressly stated Chee 200 euo 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 giagen com 2010 QIAGEN all rights reserved eee 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 Orders 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
24. f RNeasy Universal Kits is tested against predetermined specifications to ensure consistent product quality 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 QlAzol Lysis Reagent and Buffer RWT contain guanidine thiocyanate Guanidine salts can form highly reactive compounds when combined with bleach If liquid containing these buffers is spilt clean with suitable 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 196 v v sodium hypochlorite 6 RNeasy Plus Universal Handbook 09 2010 The following risk and safety phrases apply to the components of RNeasy Plus Universal Kits QIAzol Lysis Reagent Contains phenol guanidine thiocyanate toxic corrosive Risk and safety phrases R23 24 25 32 34 48 20 21 22 68 S24 25 26 36 37 39 45 gDNA Eliminator Solution Contains cetrimonium bromide dangerous for the environment Risk and safety phrases R52 53 Buffer RWT Conta
25. fication 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 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 To remove RNase contamination from bench surfaces nondisposable plasticware and laboratory equipment e g pipets and electrophoresis tanks use of RNasekKiller cat no 2500080 from 5 PRIME www 5prime com is recommended RNase contamination can alternatively be removed using general laboratory reagents To decontaminate plasticware rinse with 0 1 M NaOH 1 mM EDTA followed by RNase free water see Solutions page 32 or rinse with chloroform if the plasticware is chloroform resistant To decontaminate electrophoresis tanks clean with detergent e g 0 596 SDS rinse with RNase free water rinse with ethanol if the tanks are ethanol resistant and allow to dry Disposable plasticware
26. h quality total RNA gDNA Eliminator Solution effectively removes genomic DNA contamination eliminating the need for DNase digestion It also significantly improves separation of DNA into the interphase The RNeasy Plus Universal protocol is easy to follow and compared to phenol chloroform treatment with a subsequent cleanup procedure much faster and more efficient with better RNA purity and yield QIAGEN also provides a wide range of other kits for purification of total RNA from different sample sources visit www qiagen com RNA Principle and procedure RNeasy Plus Universal Kits integrate phenol guanidine based sample lysis and silica membrane purification of total RNA QlAzol Lysis Reagent included in the kits is a monophasic solution of phenol and guanidine thiocyanate designed to facilitate lysis of all kinds of tissues and inhibit RNases The high lysis efficiency of the reagent and the subsequent removal of contaminants by organic phase extraction enable use of larger amounts of tissue with RNeasy spin columns E RNeasy Plus Universal Mini Kit up to 50 mg of tissue or up to 100 mg of brain or adipose tissue per RNeasy Mini spin column E RNeasy Plus Universal Midi Kit up to 250 mg of tissue or up to 500 mg of brain or adipose tissue per RNeasy Midi spin column Tissue samples are homogenized in QIAzol Lysis Reagent After addition of gDNA Eliminator Solution and chloroform the homogenate is separated into aqueous and
27. he collection tube in step 13 13 Add 4 ml Buffer RWT to the RNeasy spin column Close the lid gently and centrifuge for 5 min at 3000 5000 x g to wash the membrane Discard the flow through Reuse the collection tube in step 14 Note Buffer RWT is supplied as a concentrate Ensure that ethanol is added to Buffer RWT before use see Things to do before starting page 23 14 Add 2 5 ml Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge for 2 min at 3000 5000 x g to wash the membrane Discard the flow through Reuse the collection tube in step 15 Note Buffer RPE is supplied as a concentrate Ensure that ethanol is added to Buffer RPE before use see Things to do before starting page 23 15 Add 2 5 ml Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge for 5 min at 3000 5000 x g to wash the membrane The long centrifugation dries the spin column membrane ensuring that no ethanol is carried over during RNA elution Residual ethanol may interfere with downstream reactions Note After centrifugation carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur 16 Place the RNeasy spin column in a new 15 ml collection tube supplied Add the appropriate volume of RNase free water see Table 3 page 26 directly to the spin column membrane Close the lid gently To elute the
28. icals 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 Plus Universal Handbook 09 2010 33 Concentration of RNA sample 44 ug ml x Aseo x dilution factor 44 ug ml x 0 2 x 50 440 ug ml concentration x volume in milliliters 440 ug ml x 0 1 ml 44 ug of RNA Total amount Purity of RNA The ratio of the readings at 260 nm and 280 nm A260 A280 provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV spectrum such as protein However the A A gg ratio is influenced considerably by pH Since water is not buffered the pH and the resulting Ax60 A280 ratio can vary greatly Lower pH results in a lower A260 A2g0 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 A260 A280 ratio of 1 9 2 1 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 Asso reading of 1 44 ug ml RNA is based on an extinction coefficient calculated for RNA at neutral pH see Spectrophotometric quantification of RNA page 33 DNA contam
29. ination No currently available purification method can guarantee that RNA is completely free of DNA even when it is not visible on an agarose gel RNeasy Kits will however remove the vast majority of cellular DNA gDNA Eliminator Solution helps to further reduce genomic DNA contamination but trace amounts of genomic DNA may still remain depending on the amount and nature of the sample For analysis of very low abundance targets any interference by residual DNA contamination can be detected by performing real time RT PCR control experiments in which no reverse transcriptase is added prior to the PCR step Wilfinger W W Mackey M and Chomczynski 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 EEEEEuuuuuuuutnt o uL L LL Au LA 0 ER 34 RNeasy Plus Universal Handbook 09 2010 To prevent any interference by DNA in real time RT PCR applications such as with Applied Biosystems and Rotor Gene instruments we recommend designing primers that anneal at intron splice junctions 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 see www giagen com GeneGlobe For real time RT PCR assays where amp
30. ins 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 R23 24 25 Toxic by inhalation in contact with skin and if swallowed R32 Contact with acids liberates very toxic gas R34 Causes burns R48 20 21 22 Harmful danger of serious damage to health by prolonged exposure through inhalation in contact with skin and if swallowed R52 53 Harmful to aquatic organisms and may cause long term adverse effects to the aquatic environment R68 Possible risks of irreversible effects S13 Keep away from food drink and animal feeding stuffs S24 25 Avoid contact with skin and eyes S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S36 Wear suitable protective clothing 836 37 39 Wear suitable protective clothing gloves and eye face protection S45 In case of accident or if you feel unwell seek medical advice immediately show the label where possible S46 If swallowed seek medical advice immediately and show container or label RNeasy Plus Universal Handbook 09 2010 7 Introduction RNeasy Plus Universal Kits are designed for lysis of all kinds of tissues and subsequent purification of hig
31. ion and homogenization than conical bottomed tubes E Place the tip of the disposable probe into the vessel and operate the TissueRuptor at full speed until the lysate is uniformly homogeneous usually 20 40 s Proceed to step 4 Note To avoid damage to the TissueRuptor and disposable probe during operation make sure the tip of the probe remains submerged in the buffer Foaming may occur during homogenization especially of brain tissue If this occurs let the homogenate stand at room temperature for 2 3 min until the foam subsides before continuing with the procedure 3b Disruption and homogenization using the TissueLyser LT E Keep the tubes prepared in step 1 on dry ice for at least 15 min however keep the insert of the TissueLyser LT Adapter at room temperature Then place the tissues in the tubes and keep the tubes on dry ice for another 15 min If working with RNAlater or Allprotect stabilized tissues it is not necessary to place the tubes on dry ice E Place the tubes in the insert of the TissueLyser LT Adapter and incubate at room temperature for 2 min Then immediately add 900 ul QlAzol Lysis Reagent per tube Do not incubate for longer than 2 min otherwise frozen tissues will thaw resulting in potential RNA degradation E Place the tubes in the TissueLyser LT Adapter 18 RNeasy Plus Universal Handbook 09 2010 E Operate the TissueLyser LT for 2 5 min at 50 Hz The time depends on the tissue being processe
32. is step to eliminate any possible carryover of Buffer RPE or if residual flow through remains on the outside of the RNeasy spin column after step C C8 Place the RNeasy spin column in a new 1 5 ml collection tube supplied Add 30 50 ul RNase free water directly to the spin column membrane Close the lid gently To elute the RNA centrifuge for 1 min at 28000 x g 210 000 rpm C9 Repeat step C8 using another volume of RNase free water or using the eluate from step C8 if high RNA concentration is required Reuse the collection tube from step C8 If using the eluate from step C8 the RNA yield will be 15 30 less than that obtained using a second volume of RNase free water but the final RNA concentration will be higher RNeasy Plus Universal Handbook 09 2010 37 Appendix D Purification of Total RNA Containing miRNA Using the RNeasy Plus Universal Midi Kit The RNeasy Plus Universal Midi Kit can also be used to purify total RNA that contains small RNAs such as miRNA Procedure Follow the protocol on pages 22 24 up to and including step 9 and then follow steps D1 D8 below D1 Add 1 5 volumes usually 4 5 ml of 100 ethanol and mix thoroughly by pipetting up and down Do not centrifuge Proceed immediately to step D2 Note The volume of lysate may be less than 4 5 ml due to loss during homogenization and centrifugation Precipitates may be visible after addition of ethanol Resuspend precipitates completely by vigorou
33. ization of RNA in tissues see page 13 RNAlater RNA Stabilization Reagent or Allprotect Tissue Reagent see ordering information page 40 or liquid nitrogen and dry ice Users of RNeasy Plus Universal Mini Kit BW 1 5 ml or 2 ml microcentrifuge tubes M Microcentrifuge s with rotor for 2 ml tubes for centrifugation at 4 C and at room temperature 15 25 C M Equipment for tissue disruption and homogenization see page 14 we recommend the TissueRuptor the TissueLyser LT or the TissueLyser II see ordering information page 41 Users of RNeasy Plus Universal Midi Kit E 10 15 ml centrifuge tubes E Laboratory centrifuge s capable of 5000 x g for centrifugation at 4 C and at room temperature 15 25 C M Equipment for tissue disruption and homogenization see page 14 we recommend the TissueRuptor see ordering information page 41 Do not use denatured alcohol which contains other substances such as methanol or methylethylketone t All centrifugation steps are carried out in a conventional laboratory centrifuge e g QIAGEN 96 Well Plate Centrifugation System please inquire with a swinging bucket rotor for 15 ml centrifuge tubes The maximum speed of 3500 5000 rpm corresponds to 3000 5000 x g for most rotors RNeasy Midi spin columns fit into 15 ml collection tubes supplied with the kit These fit into the rotor of almost every standard laboratory centrifuge available In the unlikely event that the tubes do not fit RNeas
34. lable at www giagen com or can be requested from QIAGEN Technical Services or your local distributor ER 42 RNeasy Plus Universal Handbook 09 2010 Trademarks QIAGEN QlAxcel QlAzol GeneGlobe QuantiTect RNeasy TissueRuptor QIAGEN Group Agilent Agilent Technologies Inc Applied Biosystems Applera Corporation Rotor Gene Corbett Research Pty Ltd SYBR Molecular Probes Inc RNAlater is a trademark of AMBION Inc Austin Texas and is covered by various U S and foreign patents Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the RNeasy Plus Universal Mini and Midi Kits to the following terms 1 The RNeasy Plus Universal Mini and Midi Kits may be used solely in accordance with the RNeasy Plus Universal 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 Plus Universal 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 components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied oth
35. lification of genomic DNA cannot be avoided we recommend using the QuantiTect Reverse Transcription Kit for reverse transcription The kit integrates fast cDNA synthesis with rapid removal of genomic DNA contamination see ordering information page 42 Integrity of RNA The integrity and size distribution of total RNA purified with RNeasy Plus Universal Kits can be checked by denaturing agarose gel electrophoresis and ethidium bromide staining or by using the QlAxcel system or 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 sample suffered major degradation either before or during RNA purification The Agilent 2100 Bioanalyzer also provides an RNA Integrity Number RIN as a useful measure of RNA integrity Ideally the RIN should be close to 10 but in many cases particularly with tissue samples RNA quality is greatly influenced by how well the original sample was preserved 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 Plus Universal Handbook 09 2010 35 Appendix C Purification of Total RNA Contai
36. load the RNeasy spin column as this will significantly reduce RNA yield and quality Weighing tissue is the most accurate way to quantify the amount of starting material As a guide a 4 mm cube 64 mm of most animal tissues weighs 70 85 mg Important points before starting E If using RNeasy Plus Universal Kits for the first time read Important Notes page 12 W If working with RNA for the first time read Appendix A page 31 E f using a TissueRuptor or TissueLyser system ensure that you are familiar with operating it by referring to the supplied user manual operating instructions and handbook M To freeze tissue for long term storage several months flash freeze in liquid nitrogen and immediately transfer to 70 C Do not allow tissues to thaw during weighing or handling prior to disruption in QlAzol Lysis Reagent Homogenized tissue lysates from step 3 can also be stored at 70 C for several months Incubate frozen lysates at 37 C in a water bath until completely thawed and salts are dissolved before continuing with step 4 Avoid prolonged incubation which may compromise RNA integrity E Generally DNase digestion is not required since integrated QlAzol gDNA Eliminator Solution and RNeasy technologies efficiently remove most of the genomic DNA contamination without DNase treatment For real time two 16 RNeasy Plus Universal Handbook 09 2010 step RT PCR applications further DNA removal can be achieved using the
37. lumn Maximum binding capacity 100 ug RNA 1 mg RNA Maximum loading volume 700 ul 4 ml RNA size distribution RNA gt 200 RNA gt 200 nucleotides nucleotides Minimum elution volume 30 ul 150 ul Maximum amount of starting lt 50 mg lt 250 mg tissue Note If the binding capacity of the RNeasy spin column is exceeded RNA yields will not be consistent and may be reduced If lysis of the starting material is incomplete RNA yields will be lower than expected even if the binding capacity of the RNeasy spin column is not exceeded FERRE LLLI ee 12 RNeasy Plus Universal Handbook 09 2010 Table 2 Typical yields of total RNA with RNeasy Plus Universal Kits Mouse rat tissue 10 mg Yield of total RNA ug Adipose tissue 0 5 2 5 Brain 5 20 Heart 5 25 Intestine 10 60 Kidney 5 40 Liver 15 80 Lung 5 15 Muscle 5 35 Skin 2 5 Spleen 15 100 Amounts can vary due to factors such as species and developmental stage especially with adipose tissues large variations are possible due to developmental stage and location of the tissue Since the RNeasy procedure enriches for mRNA and other RNA species 7 200 nucleotides the total RNA yield does not include 5S rRNA tRNA and other low molecular weight RNAs which make up 15 2096 of total cellular RNA Handling and storing starting material RNA in harvested tissue is not protected until the sample is treated with RNAlater RNA Stabilization Reagent flash frozen or disrupted
38. n steps should be performed at room temperature 15 25 C During the procedure work quickly Things to do before starting E Buffer RWT is supplied as a concentrate Before using for the first time add 2 volumes of ethanol 96 100 as indicated on the bottle to obtain a working solution Buffer RPE is supplied as a concentrate Before using for the first time add 4 volumes of ethanol 96 10096 as indicated on the bottle to obtain a working solution Procedure 1 Excise the tissue sample from the animal or remove it from storage Determine the amount of tissue Do not use more than 250 mg tissue or more than 500 mg brain or adipose tissue Proceed immediately to step 2 Weighing tissue is the most accurate way to determine the amount If the tissue sample was stored in RNAlater or Allprotect Reagent remove it from the reagent using forceps and be sure to remove any excess reagent or crystals that may have formed RNA in harvested tissues is not protected until the tissues are treated with RNAlater or Allprotect Reagent flash frozen or disrupted and homogenized in step 3 Frozen tissues should not be allowed to thaw during handling The relevant procedures should be carried out as quickly as possible 2 Place the tissue in a suitably sized vessel containing 5 ml QIAzol Lysis Reagent Note Use a suitably sized vessel with sufficient extra headspace to accommodate foaming which may occur during homogenization
39. neral remarks on handling RNA Do not put RNA samples into a vacuum dryer that has been used in DNA preparation where RNases may have been used DNA contamination in downstream experiments a Phase separation The phase separation step 8 should be performed at too high performed at 4 C to allow optimal phase a temperature separation and removal of genomic DNA from the aqueous phase Make sure that the centrifuge does not heat above 10 C during the centrifugation b Interphase Contamination of the aqueous phase with the contamination of interphase results in an increased DNA content aqueous phase in the RNA eluate Make sure to transfer the aqueous phase without interphase contamination RNeasy Plus Universal Handbook 09 2010 29 E S Not enough QlAzol Lysis Reagent used for homogenization Organic solvents in samples used for RNA purification No gDNA Eliminator Solution added before phase separation Lysate mixed with chloroform by vortexing Comments and suggestions In subsequent preparations reduce the amount of starting material and or increase the volume of QIAzol Lysis Reagent and the homogenization time Make sure that the starting sample does not contain organic solvents e g ethanol DMSO strong buffers or alkaline reagents These can interfere with the phase separation In subsequent preparations add the appropriate amount of gDNA Eliminator Solution Mix lysate and chloroform b
40. ning miRNA Using the RNeasy Plus Universal Mini Kit The RNeasy Plus Universal Mini Kit can also be used to purify total RNA that contains small RNAs such as miRNA Procedure Follow the protocol on pages 16 20 up to and including step 9 and then follow steps C1 C9 below C1 Add 1 5 volumes usually 900 pl of 100 ethanol and mix thoroughly by pipetting up and down Do not centrifuge Proceed immediately to step C2 Note The volume of lysate may be less than 600 ul due to loss during homogenization and centrifugation Precipitates may be visible after addition of ethanol Resuspend precipitates completely by vigorous shaking and proceed immediately to step C2 C2 Transfer up to 700 pl of the sample to an RNeasy Mini 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 at room temperature 15 25 C Discard the flow through Reuse the collection tube in step C3 C3 Repeat step C2 using the remainder of the sample Discard the flow through Reuse the collection tube in step C4 C4 Add 700 pl Buffer RWT to the RNeasy spin column Close the lid gently and centrifuge for 15 s at 28000 x g 210 000 rpm to wash the membrane Discard the flow through Reuse the collection tube in step C5 After centrifugation carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow through Be sure to empty
41. ns Phases do not separate completely a No chloroform added Make sure to add chloroform that does not or chloroform not pure contain isoamyl alcohol or other additives b Homogenate not After addition of chloroform step 6 the sufficiently mixed homogenate must be vigorously shaken If the before centrifugation phases are not well separated shake the tube vigorously for at least 15 s and repeat the incubation and centrifugation in steps 7 and 8 c Organic solvents in Make sure that the starting sample does not samples used for RNA contain organic solvents e g ethanol DMSO purification strong buffers or alkaline reagents These can interfere with the phase separation Clogged RNeasy spin column a Inefficient disruption See Disrupting and homogenizing starting and or material page 14 for details on disruption and homogenization homogenization methods Increase g force and centrifugation time if necessary In subsequent preparations reduce the amount of starting material see page 12 and protocol page 16 or 22 and or increase the homogenization time b Too much starting In subsequent preparations reduce the amount material of starting material It is essential to use the correct amount of starting material see page 12 and protocol page 16 or 22 RNeasy Plus Universal Handbook 09 2010 27 c Centrifugation temperature too low Low RNA yield a Insufficient disruption and homogenization b T
42. ometric quantification of RNA below For small amounts of RNA however it may be difficult to determine amounts photometrically Small amounts of RNA can be quantified using the QlAxcel system www giagen com QlAxcel or Agilent 2100 Bioanalyzer quantitative RT PCR or fluorometric quantification Spectrophotometric quantification of RNA To ensure significance Asso readings should be greater than 0 15 An absorbance of 1 unit at 260 nm corresponds to 44 ug of RNA per ml Agg9 1 44 ug 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 34 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 32 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 100 ul Dilution 10 ul of RNA sample 490 ul of 10 mM Tris Cl pH 7 0 1 50 dilution Measure absorbance of diluted sample in a 1 ml cuvette RNase free 0 0 2 When working with chem
43. oo much starting material c RNA still bound to RNeasy spin column membrane d Centrifugation temperature too low Comments and suggestions Except for phase separation step 8 all centrifugation steps should be performed at 15 25 C Some centrifuges may cool to below 20 C even when set at 20 C This can cause formation of precipitates that can clog the RNeasy spin column If this happens set the centrifugation temperature to 25 C Warm the ethanol containing lysate to 37 C before transferring to the RNeasy spin column See Disrupting and homogenizing starting material page 14 for details on disruption and homogenization methods In subsequent preparations reduce the amount of starting material It is essential to use the correct amount of starting material see page 12 and protocol page 16 or 22 Repeat RNA elution but incubate the RNeasy spin column on the benchtop for 10 min with RNase free water before centrifuging Except for phase separation step 8 all centrifugation steps should be performed at 15 25 C Some centrifuges may cool to below 20 C even when set at 20 C This can cause formation of precipitates that can clog the RNeasy spin column If this happens set the centrifugation temperature to 25 C Warm the ethanol containing lysate to 37 C before transferring to the RNeasy spin column Low or no recovery of RNA RNeasy Plus Universal Mini Kit RNase free water incorrectly dispensed
44. organic phases by centrifugation RNA partitions to the upper aqueous phase while DNA partitions to the interphase and proteins to the lower organic phase or the interphase The upper aqueous phase is collected and RNA is purified using RNeasy spin columns The steps of the RNA purification procedure differ depending on the kit format The upper aqueous phase is mixed with ethanol to provide appropriate binding conditions and applied to an RNeasy Mini spin column Total RNA binds to the spin column membrane and phenol and other contaminants are efficiently washed away High quality RNA is then eluted in RNase free water see flowchart page 10 The standard protocols for RNeasy Plus Universal Kits pages 16 and 22 allow purification of all RNA molecules longer than 200 nucleotides The procedure provides an enrichment for mRNA since most RNAs 200 nucleotides such as 5 8S rRNA 5S rRNA and tRNAs which together comprise 15 2096 of total To ensure optimal RNA yields the binding capacity of the RNeasy spin column must not be exceeded For details see the individual protocols 8 RNeasy Plus Universal Handbook 09 2010 RNA are selectively excluded The size distribution of the purified RNA is comparable to that obtained by centrifugation through a CsCl gradient or cushion where small RNAs do not sediment efficiently For purification of total RNA containing small RNAs such as microRNA miRNA using the RNeasy Plus Universal Mini
45. ormation 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 QIAGEN products If you have any questions or experience any difficulties regarding RNeasy Plus Universal Kits or QIAGEN products in general please do not hesitate to contact us RNeasy Plus Universal Handbook 09 2010 5 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 qiagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www qiagen com Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot o
46. otes page 12 W If working with RNA for the first time read Appendix A page 31 E f using the TissueRuptor ensure that you are familiar with operating it by referring to the TissueRuptor User Manual and TissueRuptor Handbook M To freeze tissue for long term storage several months flash freeze in liquid nitrogen and immediately transfer to 70 C Do not allow tissues to thaw during weighing or handling prior to disruption in QlAzol Lysis Reagent Homogenized tissue lysates from step 3 can also be stored at 70 C for several months Incubate frozen lysates at 37 C in a water bath until completely thawed and salts are dissolved before continuing with step 4 Avoid prolonged incubation which may compromise RNA integrity E Generally DNase digestion is not required since integrated QlAzol gDNA Eliminator Solution and RNeasy technologies efficiently remove most of the DNA without DNase treatment For real time two step RT PCR applications 22 RNeasy Plus Universal Handbook 09 2010 further DNA removal can be achieved using the QuantiTect Reverse Transcription Kit which provides cDNA synthesis with integrated removal of genomic DNA contamination see ordering information page 42 M QlAzol Lysis Reagent and Buffer RWT contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach See page 6 for safety information E Except for phase separation step 8 all protocol and centrifugatio
47. ppendix B Storage Quantification and Determination of Quality of RNA 33 Appendix C Purification of Total RNA Containing miRNA Using the RNeasy Plus Universal Mini Kit 36 Appendix D Purification of Total RNA Containing miRNA Using the RNeasy Plus Universal Midi Kit 38 Ordering Information 40 RNeasy Plus Universal Handbook 09 2010 3 Kit Contents RNeasy Plus Universal Kit Mini 50 Midi 10 Catalog no 73404 73442 Number of preps 50 10 RNeasy Mini Spin Columns pink 50 each in a 2 ml Collection Tube RNeasy Midi Spin Columns pink 10 each in a 15 ml Collection Tube Collection Tubes 1 5 ml 50 Collection Tubes 2 ml 50 Collection Tubes 15 ml 10 QlAzol Lysis Reagent 50 ml 50 ml gDNA Eliminator Solution 8 ml 8 ml Buffer RWT concentrate 15 ml 15 ml Buffer RPE concentrate 11 ml 11 ml RNase Free Water 10 ml 10 ml Handbook Contains a guanidine salt Not compatible with disinfectants containing bleach See page 6 for safety information t Buffer RWT is supplied as a concentrate Before using for the first time add 2 volumes of ethanol 96 100 as indicated on the bottle to obtain a working solution 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 Note QlAzol Lysis Reagent is delivered separately 4 RNeasy Plus Universal Handbook 09 2010 Storage RNeasy
48. roducts Allprotect Tissue Reagent 100 ml RNAlater RNA Stabilization Reagent 50 ml RNAlater RNA Stabilization Reagent 250 ml RNAlater TissueProtect Tubes 50 x 1 5 ml RNAlater TissueProtect Tubes 20 x 5 ml QIAzol Lysis Reagent 200 ml RNeasy 96 Universal Tissue Kit 4 40 Contents For 50 RNA minipreps RNeasy Mini Spin Columns gDNA Eliminator Solution Collection Tubes RNase Free Water and Buffers For 10 RNA midipreps RNeasy Midi Spin Columns gDNA Eliminator Solution Collection Tubes RNase Free Water and Buffers For stabilization of DNA RNA and protein in 50 x 200 mg tissue samples 100 ml Allprotect Tissue Reagent Allprotect Reagent Pump For stabilization of RNA in 25 x 200 mg tissue samples 50 ml RNAlater RNA Stabilization Reagent For stabilization of RNA in 125 x 200 mg tissue samples 250 ml RNAlater RNA Stabilization Reagent For stabilization of RNA in 50 x 150 mg tissue samples 50 screw top tubes containing 1 5 ml RNAlater RNA Stabilization Reagent each For stabilization of RNA in 20 x 500 mg tissue samples 20 screw top tubes containing 5 ml RNAlater RNA Stabilization Reagent each 200 ml QIAzol Lysis Reagent For 4 x 96 total RNA preps 4 RNeasy 96 Plates Collection Microtubes Elution Microtubes CL Caps S Blocks AirPore Tape Sheets QIAzol Lysis Reagent RNase Free Reagents and Buffers Cat no 73404 73442 76405 76104 76106 76154 76163
49. s shaking and proceed immediately to step D2 D2 Transfer up to 4 ml of the sample to an RNeasy Midi 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 at room temperature 15 25 C Discard the flow through Reuse the collection tube in step D3 D3 Repeat step D2 using the remainder of the sample Discard the flow through Reuse the collection tube in step D4 D4 Add 4 ml Buffer RWT to the RNeasy spin column Close the lid gently and centrifuge for 5 min at 3000 5000 x g to wash the membrane Discard the flow through Reuse the collection tube in step D5 Note Buffer RWT is supplied as a concentrate Ensure that ethanol is added to Buffer RWT before use see Things to do before starting page 23 D5 Add 2 5 ml Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge for 2 min at 3000 5000 x g to wash the membrane Discard the flow through Reuse the collection tube in step D Note Buffer RPE is supplied as a concentrate Ensure that ethanol is added to Buffer RPE before use see Things to do before starting page 23 Flow through contains QIAzol Lysis Reagent or Buffer RWT and is therefore not compatible with bleach See page 6 for safety information 38 RNeasy Plus Universal Handbook 09 2010 D6 Add 2 5 ml Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge for 5 min at 3000 5000 x g to w
50. ssues can be disrupted by rapid agitation in the presence of beads and lysis buffer Disruption and simultaneous homogenization occur by the shearing and crushing action of the beads as they collide with the cells Two bead mills are available from QIAGEN the TissueLyser LT for low to medium throughput disruption and the TissueLyser Il for medium to high throughput disruption The TissueLyser LT disrupts and homogenizes up to 12 samples at the same time The instrument needs to be used in combination with the TissueLyser LT Adapter which holds 12 x 2 ml microcentrifuge tubes containing stainless steel beads of 5 mm or 7 mm mean diameter For guidelines on using the TissueLyser LT refer to the TissueLyser LT Handbook BERAAALLLLLLLLLLLLLLLLLL LLEN 14 RNeasy Plus Universal Handbook 09 2010 The TissueLyser ll disrupts and homogenizes up to 48 tissue samples simultaneously when used in combination with the TissueLyser Adapter Set 2 x 24 which holds 48 x 2 ml microcentrifuge tubes containing stainless steel beads of 5 mm mean diameter For guidelines on using the TissueLyser II refer to the TissueLyser Handbook If using other bead mills for sample disruption and homogenization refer to suppliers guidelines Note Tungsten carbide beads react with QIAzol Lysis Reagent and must not be used to disrupt and homogenize tissues The TissueLyser ll can also disrupt and homogenize up to 192 tissue samples simultaneously when used in
51. the collection tube completely Note Buffer RWT is supplied as a concentrate Ensure that ethanol is added to Buffer RWT before use see Things to do before starting page 17 Flow through contains QIAzol Lysis Reagent or Buffer RWT and is therefore not compatible with bleach See page 6 for safety information 36 RNeasy Plus Universal Handbook 09 2010 C5 Add 500 ul Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge for 15 s at 28000 x g 210 000 rpm to wash the membrane Discard the flow through Reuse the collection tube in step C Note Buffer RPE is supplied as a concentrate Ensure that ethanol is added to Buffer RPE before use see Things to do before starting page 17 C6 Add 500 ul Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge for 2 min at 28000 x g 210 000 rpm to wash the membrane The long centrifugation dries the spin column membrane ensuring that no ethanol is carried over during RNA elution Residual ethanol may interfere with downstream reactions Note After centrifugation carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur C7 Optional Place the RNeasy spin column in a new 2 ml collection tube supplied and discard the old collection tube with the flow through Close the lid gently and centrifuge at full speed for 1 min Perform th
52. urther treatment with DNase unnecessary 6 Add 180 ul chloroform Securely cap the tube containing the homogenate and shake it vigorously for 15 s Thorough mixing is important for subsequent phase separation 7 Place the tube containing the homogenate on the benchtop at room temperature for 2 3 min RNeasy Plus Universal Handbook 09 2010 19 8 Centrifuge at 12 000 x g for 15 min at 4 C After centrifugation heat the centrifuge to room temperature 15 25 C if the same centrifuge will be used in the later steps of this procedure After centrifugation the sample separates into 3 phases an upper colorless aqueous phase containing RNA a white interphase and a lower red organic phase For tissues with an especially high fat content an additional clear phase may be visible below the red organic phase The volume of the aqueous phase should be approximately 600 ul 9 Transfer the upper aqueous phase usually 600 pl to a new microcentrifuge tube not supplied 10 Add 1 volume usually 600 pl of 70 ethanol and mix thoroughly by pipetting up and down Do not centrifuge Proceed immediately to step 11 Note The volume of lysate may be less than 600 ul due to loss during homogenization and centrifugation Precipitates may be visible after addition of ethanol Resuspend precipitates completely by vigorous shaking and proceed immediately to step 11 11 Transfer up to 700 pl of the sample to an RNeasy Mini spin column plac
53. y Midi spin columns can also be inserted into different 12 15 ml RNase free glass or polypropylene tubes PE EER RNeasy Plus Universal Handbook 09 2010 11 Important Notes Determining the amount of starting material It is essential to use the correct amount of starting material in order to obtain optimal RNA yield and purity The maximum amount that can be used is determined by E The type of tissue and its RNA content E The volume of QIAzol Lysis Reagent required for efficient lysis M The RNA binding capacity of the RNeasy spin column When processing samples containing high amounts of RNA less than the maximum amount of starting material shown in Table 1 should be used so that the RNA binding capacity of the RNeasy spin column is not exceeded When processing samples containing low amounts of RNA the maximum amount of starting material shown in Table 1 can be used However even though the RNA binding capacity of the RNeasy spin column is not reached the maximum amount of starting material must not be exceeded Otherwise lysis will be incomplete and cellular debris may interfere with the binding of RNA to the RNeasy spin column membrane resulting in lower RNA yield and purity More information on using the correct amount of starting material is given in the protocols Table 2 shows expected RNA yields from various sources Table 1 RNeasy spin column specifications RNeasy Mini spin RNeasy Midi spin Specification column co
54. y shaking vigorously Do not vortex the tubes RNA does not perform well in downstream experiments a b 30 Salt carryover during elution Ethanol carryover Ensure that Buffer RPE is at 20 30 C During the second wash with Buffer RPE be sure to dry the RNeasy spin column membrane by centrifuging at 28000 x g 210 000 rpm for 2 min at 15 25 C RNeasy Plus Universal Mini Kit step 15 of protocol or at 3000 5000 x g for 5 min at 15 25 C RNeasy Plus Universal Midi Kit step 14 of protocol After centrifugation carefully remove the column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur To eliminate any chance of possible ethanol carryover with the RNeasy Plus Universal Mini Kit place the RNeasy Mini spin column in a new 2 ml collection tube and perform the optional min centrifugation step as described in step 16 of the protocol RNeasy Plus Universal Handbook 09 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 puri
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