RNeasy® Microarray Tissue Handbook
Contents
1. PDF format at www giagen 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 QIAzol Lysis Reagent contains guanidine thiocyanate and Buffer RW1 contains a small amount of 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 1 v v sodium hypochlorite The following risk and safety phrases apply to the components of the RNeasy Microarray Tissue Mini Kit QlAzol 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 Buffer RW1 Contains ethanol flammable Risk phrase R10 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 R10 Flammable 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 in2. Tissue Handbook 11 2009 Product Use Limitations The RNeasy Microarray Tissue Mini Kit is intended for molecular biology applications This product is neither intended for the diagnosis prevention or treatment of a disease nor has it been validated for such use either alone or in combination with other products Therefore the performance characteristics of this product for clinical use i e diagnostic prognostic therapeutic or blood banking are unknown 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 p
3. V 60 Hz for North America and Japan 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 32 RNeasy Microarray Tissue Handbook 11 2009 Ordering Information Product TissueRuptor Disposable Probes 25 TissueLyser LT Tissuelyser LT Adapter 12 Tube Sample Tubes RB 2 ml TissueLyser Il Tissuelyser Adapter Set 2 x 24 TissueLyser Single Bead Dispenser 5 mm Stainless Steel Beads 5 mm 200 Stainless Steel Beads 7 mm 200 RNase Free DNase Set 50 Collection Tubes 2 ml Tissuelyser Adapter Set 2 x 96 Collection Microtubes racked Contents 25 nonsterile plastic disposable probes for use with the TissueRuptor Compact bead mill 100 240 V AC 50 60 Hz requires the TissueLyser LT Adapter 12 Tube available separately Adapter for disruption of up to 12 samples in 2 ml microcentrifuge tubes on the TissueLyser LT 1000 safe lock microcentrifuge tubes 2 ml for use with the TissueLyser LT Bead mill 100 120 220 240 V 50 60 Hz requires the TissueLyser Adapter Set 2 x 24 or TissueLyser Adapter Set 2 x 96 available separately 2 sets of Adapter Plates and 2 racks for use with 2 ml microcentrifuge tubes on the TissueLyser Il For dispensing individual beads 5 mm diameter Stainless Steel Beads suitable for use with TissueLyser systems Stainless Steel Beads suitable for use with TissueLyser syst
4. 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 Add RNase free water to the center of the RNeasy spin column membrane to ensure that the membrane is completely covered RNeasy Microarray Tissue Handbook 11 2009 Comments and suggestions Low Aggo Aogo Value a Not enough QIAzol Lysis Reagent used for homogenization b Sample not incubated for 5 min after homogenization c Water used to dilute RNA for A260 A280 Measurement RNA degraded a Inappropriate handling of starting material b RNase contamination In subsequent preparations reduce the amount of starting material and or increase the volume of QIAzol Lysis Reagent and the homogenization time Place the sample at room temperature 15 25 C for 5 min after homogenization as indicated in the protocol step 4 This step is important to promote dissociation of nucleoprotein complexes Use 10 mM Tris Cl pH 7 5 not RNase free water to dilute the sample before measuring purity see Appendix B page 27 For frozen tissue samples ensure that they were flash frozen immediately in liquid nitrogen and properly stored at 70 C Perform the RNeasy proc
5. harvested tissue is not protected until the sample is treated with RNAlater RNA Stabilization Reagent flash frozen or disrupted 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 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 12 RNeasy Microarray Tissue Handbook 11 2009 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 E Disruption Complete disruption of plasma membranes of cells and organelles is ab
6. high quality RNA For more information about the automated procedure see the relevant protocol sheet available at www qiagen com MyQlAcube 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 The QlAcube 8 RNeasy Microarray Tissue Handbook 11 2009 RNeasy Microarray Tissue Procedure Tissue Lyse and homogenize Add chloroform and shake Separate phases Add ethanol to aqueous phase Bind total RNA Wash Elute ath 9 cath 9 cat E lt t Total RNA RNeasy Microarray Tissue Handbook 11 2009 9 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 Chloroform Ethanol 70 and 96 100 Sterile RNase free pipet tips Equipment for tissue disruption and homogenization see page 13 we recommend either the TissueRuptor with TissueRuptor Disposable Probes or a Tissuelyser system see ordering information pages 32 33 For stabilization of RNA in tissues see page 12 RNAlater RNA Stabilization Reagent or Allprotect Tissue Reagent see
7. ordering information page 32 or liquid nitrogen and dry ice 1 5 ml or 2 ml microcentrifuge tubes Microcentrifuge s with rotor for 2 ml tubes for centrifugation at 4 C and at room temperature 15 25 C Do not use denatured alcohol which contains other substances such as methanol or methylethylketone 10 RNeasy Microarray Tissue Handbook 11 2009 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 E 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
8. with Buffer RW1 Washing with Buffer RPE and elution of RNA are then performed according to the standard protocol Important points before starting E Generally DNase digestion is not required since integrated QIAzol and RNeasy technologies efficiently remove most of the DNA without DNase treatment E Do not vortex the reconstituted DNase DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the tube 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 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 Prepare and load samples onto the RNeasy spin column as indicated in steps 1 10 of the protocol on pages 15 19 Instead of performing step 11 follow steps C1 C4 below 30 RNeasy Microarray Tissue Handbook 11 2009 Cl C2 C3 c4 Add 350 pl Buffer RW1 to the RNeasy spin column Close the lid gently and cent
9. 922 Technical 01293 422 999 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN anges Sample amp Assay Technologies
10. DNase treatment If desired residual DNA can be removed by optional on column DNase digestion using the RNase Free DNase Set see Appendix C page 30 RNeasy Microarray Tissue Handbook 11 2009 15 020 01d M QlAzol Lysis Reagent and Buffer RW1 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 7 all protocol and centrifugation steps should be performed at room temperature 15 25 C During the procedure work quickly Yv 2 e a 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 If performing optional on column DNase digestion prepare DNase stock solution as described in Appendix C page 30 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 RNAlafer or Allprotect Reagent place the tubes on dry ice 2 Excise the tissue sample from the animal or remove it from storage Determine the amount of tissue Do not use more than 100 mg Proceed immediately to step 3 Weighing tissue is the most accurate way to determine the amount If the tissue sample was stored in RNAlater or Allprotect Reagen
11. Frequently Asked Questions page at our Technical Support Center www qiagen 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 protocol in this handbook or sample and assay technologies for contact information see back cover or visit www giagen com Comments and suggestions Phases do not separate completely a No chloroform added or chloroform not pure b Homogenate not sufficiently mixed before centrifugation c Organic solvents in samples used for RNA purification Clogged RNeasy spin column a Inefficient disruption and or homogenization b Too much starting material Make sure to add chloroform that does not contain isoamyl alcohol or other additives After addition of chloroform step 5 the homogenate must be vigorously shaken If the phases are not well separated shake the tube vigorously for at least 15 s and repeat the incubation and centrifugation in steps 6 and 7 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 See Disrupting and homogenizing starting material page 13 for details on disruption and homogenization methods Increase gforce and centrifugation time if necessary In subsequent preparations reduce the amount of starting material see page 11 and p
12. NA from Tissues 15 Troubleshooting Guide 21 Appendix A General Remarks on Handling RNA 25 Appendix B Storage Quantification and Determination of Quality of RNA 27 Appendix C Optional On Column DNase Digestion with the RNase Free DNase Set 30 References 31 Ordering Information 32 RNeasy Microarray Tissue Handbook 11 2009 3 Kit Contents RNeasy Microarray Tissue Mini Kit 50 Catalog no 73304 Number of preps 50 RNeasy Mini Spin Columns each in a 2 ml Collection Tube 50 Collection Tubes 1 5 ml 50 Collection Tubes 2 ml 50 QIAzol Lysis Reagent 50 ml Buffer RW1 A5 ml Buffer RPE concentrate 11 ml RNase Free Water 10 ml Handbook 1 Contains a guanidine salt Not compatible with disinfectants containing bleach See page 6 for safety information Before using for the first time add 4 volumes of ethanol 6 100 as indicated on the bottle to obtain a working solution Note QIAzol Lysis Reagent is delivered separately Storage The RNeasy Microarray Tissue Mini Kit 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 Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of RNeasy Microarray Tissue Mini Kit is tested against predetermined specifications to ensure consistent product quality 4 RNeasy Microarray
13. November 2009 RNeasy Microarray Tissue Handbook For purification of total RNA from all types of tissue for microarray analysis QIAGEN 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 HM Purification of DNA RNA and proteins M Nucleic acid and protein assays E microRNA research and RNAi H 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 QIAGEN is a member of the Forest Stewardship Council FSC For the production of printed materials including handbooks QIAGEN has a policy to select suppliers that comply with FSC standards for printing processes and well managed forests Contents Kit Contents 4 Storage 4 Quality Control A Product Use Limitations 5 Product Warranty and Satisfaction Guarantee 5 Technical Assistance 5 Safety Information 6 Introduction 7 Principle and procedure 7 Automated purification 8 Equipment and Reagents to Be Supplied by User 10 Important Notes 11 Determining the amount of starting material 11 Handling and storing starting material 12 Disrupting and homogenizing starting material 13 Protocol H Purification of Total R
14. chnical Services or your local distributor Flow through contains Buffer RW1 and is therefore not compatible with bleach See page 6 for safety information RNeasy Microarray Tissue Handbook 11 2009 31 Ordering Information Product Contents Cat no RNeasy Microarray 50 RNeasy Mini Spin Columns 73304 Tissue Mini Kit 50 Collection Tubes QIAzol Lysis Reagent RNase Free Reagents and Buffers Accessories Allprotect Tissue For stabilization of DNA RNA and 76405 Reagent 100 ml protein in 50 x 200 mg tissue samples 100 ml Allprotect Tissue Reagent Allprotect Reagent Pump RNAlater RNA Stabilization For stabilization of RNA in 76104 Reagent 50 ml 25 x 200 mg tissue samples 50 ml RNAlater RNA Stabilization Reagent RNAlater RNA Stabilization For stabilization of RNA in 76106 Reagent 250 ml 125 x 200 mg tissue samples 250 ml RNAlater RNA Stabilization Reagent RNAlater TissueProtect For stabilization of RNA in 76154 Tubes 50 x 1 5 ml 50 x 150 mg tissue samples 50 screw top tubes containing 1 5 ml RNAlater RNA Stabilization Reagent each RNAlater TissueProtect For stabilization of RNA in 76163 Tubes 20 x 5 ml 20 x 500 mg tissue samples 20 screw top tubes containing 5 ml RNAlater RNA Stabilization Reagent each QIAzol Lysis Reagent 200 ml 200 ml QIAzol Lysis Reagent 79306 TissueRuptor Handheld rotor stator homogenizer 9001271 5 TissueRuptor Disposable Probes 9001272 9001273 90012745 120
15. da Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 021 3865 3865 Fax 021 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 Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 02 33430 420 Fax 02 33430 426 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 1544 7145 Fax 1544 7146 Technical 1544 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 639 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 65 67775366 Fax 65 67785177 Technical 65 67775366 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
16. e so that the column does not contact the flow through Otherwise carryover of ethanol will occur 14 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 13 Flow through contains QIAzol Lysis Reagent or Buffer RW1 and is therefore not compatible with bleach See page 6 for safety information RNeasy Microarray Tissue Handbook 11 2009 19 Y 2 o a 15 20 Place the RNeasy spin column in a new 1 5 ml collection tube supplied Add 30 50 pl RNase free water directly to the spin column membrane Close the lid gently To elute the RNA centrifuge for 1 min at gt 8000 x g 210 000 rpm Repeat step 15 using another volume of RNase free water or using the eluate from step 15 if high RNA concentration is required Reuse the collection tube from step 15 If using the eluate from step 15 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 Microarray Tissue Handbook 11 2009 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the
17. ecific 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 Microarray Tissue Handbook 11 2009 29 Appendix C Optional On Column DNase Digestion with the RNase Free DNase Set The RNase free DNase Set cat no 79254 provides efficient on column digestion of DNA during RNA purification The DNase is efficiently removed in subsequent wash steps Note Standard DNase buffers are not compatible with on column DNase digestion Use of other buffers may affect the binding of RNA to the RNeasy membrane reducing RNA yield and integrity Lysis and homogenization of the sample and binding of RNA to the RNeasy membrane are performed according to the standard protocol After washing with a reduced volume of Buffer RW1 the RNA is treated with DNase while bound to the RNeasy membrane The DNase is removed by a second wash
18. edure quickly especially the first few steps See Appendix A page 25 and Handling and storing starting material page 12 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 25 for general 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 performed at too high a temperature The phase separation step 7 should be performed at 4 C to allow optimal phase separation and removal of genomic DNA from the aqueous phase Make sure that the centrifuge does not heat above 10 C during the centrifugation RNeasy Microarray Tissue Handbook 11 2009 23 Comments and suggestions b Interphase contamination of aqueous phase c Not enough QIAzol Lysis Reagent used for homogenization d Organic solvents in samples used for RNA purification e No DNase treatment Contamination of the aqueous phase with the interphase results in an increased DNA content in the RNA eluate Make sure to transfer the aqueous phase without interphase contamination In subsequent preparations reduce the amount of starting material and or increase the volume of QIAzol Lysis Reagent and the homogenization time Make s
19. ems For 50 RNA minipreps 1500 units RNase Free DNase I RNase Free Buffer RDD and RNase Free Water 1000 x 2 ml Collection Tubes 2 sets of Adapter Plates for use with Collection Microtubes racked on the TissueLyser II Nonsterile polypropylene tubes 1 2 ml 960 in racks of 96 RNeasy Microarray Tissue Handbook 11 2009 Cat no 990890 85600 69980 990381 85300 69982 69965 69989 69990 79254 19201 69984 19560 33 Ordering Information Product Contents Collection Microtube Nonsterile polypropylene caps for Caps 120 x 8 collection microtubes 1 2 ml 960 in strips of 8 Related products RNeasy 96 Universal Tissue Kit for high throughput RNA purification from any type of animal tissue RNeasy 96 Universal For 4 x 96 total RNA preps Tissue Kit 4 4 RNeasy 96 Plates Collection Microtubes Elution Microtubes CL Caps S Blocks AirPore Tape Sheets QIAzol Lysis Reagent RNase Free Reagents and Buffers miRNeasy Kits for purification of microRNA and total RNA from a wide range of animal tissues and cells miRNeasy Mini Kit 50 For 50 preps 50 RNeasy Mini Spin Columns Collection Tubes 1 5 ml and 2 ml QIAzol Lysis Reagent RNase Free Reagents and Buffers miRNeasy 96 Kit 4 For 4 x 96 preps 4 RNeasy 96 plates Collection Microtubes racked Elution Microtubes CL Caps S Blocks AirPore Tape Sheets QIAzol Lysis Reagent RNase Free Reagents and Buffers Cat n
20. erformance 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 the RNeasy Microarray Tissue Mini 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 t 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 Microarray Tissue Handbook 11 2009 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
21. ers refer to suppliers guidelines Disruption and homogenization using TissueLyser systems In bead milling tissues 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 II for medium to high throughput disruption The TissueLyer 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 RNeasy Microarray Tissue Handbook 11 2009 13 The TissueLyser Il 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 Il 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 Il ca
22. fied directly from the aqueous phase using an RNeasy spin column No precipitation and redissolving of RNA is required which saves time prevents potential loss of RNA and avoids variability in RNA yields Principle and procedure The RNeasy Microarray Tissue Mini Kit integrates phenol guanidine based sample lysis and silica membrane purification of total RNA QIAzol Lysis Reagent included in the kit is a monophasic solution of phenol and guanidine thiocyanate designed to facilitate lysis of all types of tissue and inhibit RNases The high lysis efficiency of the reagent and the subsequent removal of contaminants by organic phase extraction enables use of larger amounts of tissue with RNeasy spin columns RNA can be purified from up to 50 mg of human or animal tissue or up to 100 mg of brain or adipose tissue per RNeasy Mini spin column Tissue samples are homogenized in QIAzol Lysis Reagent After addition of chloroform the homogenate is separated into aqueous and 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 ethanol is added to provide appropriate binding conditions The sample is then applied to an RNeasy spin column where the total RNA binds to the membrane and phenol and other contaminants are efficiently washed away High quality RNA is then eluted in RNase free
23. halation in contact with skin and if swallowed R68 Possible risk of irreversible effects 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 36 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 6 RNeasy Microarray Tissue Handbook 11 2009 Introduction The RNeasy Microarray Tissue Mini Kit provides the optimal solution for purifying RNA for microarray analysis QlAzol Lysis Reagent ensures efficient phenol guanidine based lysis of even difficultto lyse tissues while trusted RNeasy spin columns allow fast and convenient purification of intact RNA without any phenol contamination The result is highly pure RNA that can be efficiently reverse transcribed ensuring reliable results in microarray analysis and avoiding the need for unnecessary repetition of experiments The purified RNA is of the same quality as that achieved with other RNeasy Kits and is therefore also ready to use for other applications such as real time RT PCR Traditional methods of purifying RNA for microarray analysis involve acid phenol chloroform extraction followed by precipitation of RNA from the aqueous phase redissolving of the RNA and cleanup of the RNA using silica membrane technology With the RNeasy Microarray Tissue Mini Kit RNA is puri
24. ing the absorbance at 260 nm Azo in a spectrophotometer see Spectrophotometric 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 Azs readings should be greater than 0 15 An absorbance of 1 unit at 260 nm corresponds to 44 pg of RNA per ml Ayo 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 28 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 26 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 pl Dilution 10 pl of RNA sample 490 pl of 10 mM Tris Cl pH 7 0 1 50 dilution Measure absorba
25. ly 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 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 RNaseKiller 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 26 or rinse with chloroform if the plasticware is chloroform resistant To decontaminate electrophoresis tanks clean with detergent e g 0 5 SDS rinse with RNase free water rinse with ethanol if the tanks a
26. n also disrupt and homogenize up to 192 tissue samples simultaneously when used in 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 including fatty tissues in 96 well format and is based on the same technology as the RNeasy Microarray Tissue Mini Kit For ordering information see page 34 14 RNeasy Microarray Tissue Handbook 11 2009 Protocol Purification of Total RNA from Tissues 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 Microarray Tissue Mini Kit a maximum of 100 mg brain or adipose tissue can generally be processed For these tissues the RNA binding capacity of the RNeasy Mini spin column and the lysing capacity of QlAzol Lysis Reagent will not be exceeded by these amounts For other tissues a maximum of 50 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 12 If there is no information about the nature of your starting ma
27. nce of diluted sample in a 1 ml cuvette RNase free Axo 0 2 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 Microarray Tissue Handbook 11 2009 27 Concentration of RNA sample 44 pg ml x Azo x dilution factor 44 pg ml x 0 2 x 50 440 pg ml Total amount concentration x volume in milliliters 440 pg ml x 0 1 ml 44 pg of RNA Purity of RNA The ratio of the readings at 260 nm and 280 nm Ag60 Asgo provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV spectrum such as protein However the Aygo Aygo ratio is influenced considerably by pH Since water is not buffered the pH and the resulting Aoso Azgo ratio can vary greatly Lower pH results in a lower Apeo Asgo 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 Ag6o Azgo 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 Azs reading of 1 44 pg ml RNA is based on an extinction coefficient calculated fo
28. o 19566 74881 217004 217061 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 available at www giagen com or can be requested from QIAGEN Technical Services or your 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 see www giagen com RNA t Requires use of the QIAGEN 96 Well Plate Centrifugation System with refrigeration capability Tissuelyser Il recommended for disruption and homogenization QlAvac 96 optional 34 RNeasy Microarray Tissue Handbook 11 2009 Trademarks QIAGEN QlAcube QlAxcel QIAzol RNeasy TissueRuptor QIAGEN Group Agilent Agilent Technologies Inc RNAlater is a trademark of AMBION Inc Austin Texas and is covered by various U S and foreign patents 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 Microarray Tissue Mini Kit to the following terms i The RNeasy Microarray Tissue Mini Kit may be used solely in accordance with the RNeasy Microarray Tissue Handbook and for use with components contained in the Kit only QIAGEN grants no license unde
29. o 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 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 26 RNeasy Microarray Tissue Handbook 11 2009 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 measur
30. 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 Place the tissues in the tubes prepared in step 1 If the tubes were stored on dry ice place them at room temperature Then immediately add 1 ml QIAzol Lysis Reagent per tube RNeasy Microarray Tissue Handbook 11 2009 17 JO20j01q Y 2 o a m Place the tubes in the Tissuelyser Adapter Set 2 x 24 m Operate the TissueLyser Il for 2 min at 20 Hz The time depends on the tissue being processed and can be extended until the tissue is completely homogenized 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 Il for another 2 min at 20 Hz Rearranging the tubes allows even homogenization m Carefully pipet the lysates into new microcentrifuge tubes not supplied Proceed to step 4 Do not reuse the stainless steel beads 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 Add 200 pl chloroform Securely cap the tube containing the homogenate and shake it vigorously for 15 s Thorough mixing is important for subsequent phase separation Place the tube containing the homogenate on the benchtop a
31. r RNA at neutral pH see Spectrophotometric quantification of RNA page 27 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 While RNeasy Kits will remove the vast majority of cellular DNA trace amounts may still remain depending on the amount and nature of the sample If desired DNase digestion of the isolated RNA with RNase free DNase can be performed to remove residual DNA A protocol for optional on column DNase digestion using the RNase free DNase Set is provided in Appendix C page 30 The DNase is efficiently washed away in subsequent wash steps 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 28 RNeasy Microarray Tissue Handbook 11 2009 Integrity of RNA The integrity and size distribution of total RNA purified with the RNeasy Microarray Tissue Mini Kit 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 sp
32. r 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 Microarray Tissue 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 other than those expressly stated 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 2009 QIAGEN all rights reserved www qiagen com Australia Orders 03 9840 9800 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 Cana
33. re ethanol resistant and allow to dry 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 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 Microarray Tissue Handbook 11 2009 25 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 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 t
34. rifuge for 15 s at gt 8000 x g 210 000 rpm to wash the membrane Discard the flow through Reuse the collection tube in step C4 Add 10 pl DNase stock solution see above to 70 pl Buffer RDD Mix by gently inverting the tube and centrifuge briefly to collect residual liquid from the sides of the tube Buffer RDD is supplied with the RNase Free DNase Set Note DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the tube Do not vortex Add the DNase incubation mix 80 pl directly to the RNeasy spin column membrane and place on the benchtop 20 30 C for 15 min Note Be sure to add the DNase incubation mix directly to the RNeasy spin column membrane DNase digestion will be incomplete if part of the mix sticks to the walls or the O ring of the spin column Add 350 pl Buffer RW1 to the RNeasy spin column Close the lid gently and centrifuge for 15 s at gt 8000 x g 210 000 rpm Discard the flow through Continue with step 12 of the protocol on page 19 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 Te
35. rotocol page 15 and or increase the homogenization time In subsequent preparations reduce the amount of starting material It is essential to use the correct amount of starting material see page 11 and protocol page 15 RNeasy Microarray Tissue Handbook 11 2009 21 Comments and suggestions c Centrifugation temperature too low Low RNA yield a Insufficient disruption and homogenization b Too much starting material c RNA still bound to RNeasy spin column membrane d Centrifugation temperature too low Low or no recovery of RNA 22 RNase free water incorrectly dispensed Except for phase separation step 7 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 13 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 11 and protocol page 15 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 7
36. solutely required to release all the RNA contained in the sample Incomplete disruption results in significantly reduced RNA yields MH 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 homogeniz
37. starting material is given in the protocol Table 2 shows expected RNA yields from various sources Table 1 RNeasy spin column specifications Specification RNeasy Mini spin column Maximum binding capacity 100 pg RNA Maximum loading volume 700 pl RNA size distribution RNA gt 200 nucleotides Minimum elution volume 30 pl Maximum amount of starting tissue lt 100 mg 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 RNeasy Microarray Tissue Handbook 11 2009 11 Table 2 Typical yields of total RNA with the RNeasy Microarray Tissue Mini Kit Mouse rat tissue 10 mg Yield of total RNA yg 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 gt 200 nucleotides the total RNA yield does not include 5S rRNA tRNA and other low molecular weight RNAs which make up 15 20 of total cellular RNA Handling and storing starting material RNA in
38. t 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 pro cedures should be carried out as quickly as possible 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 13 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 When disrupting tough or very tough samples with the TissueLyser LT we recommend using one or two 7 mm stainless steel beads 16 RNeasy Microarray Tissue Handbook 11 2009 3a Disruption and homogenization using the TissueRuptor Place the tissue in a suitably sized vessel containing 1 ml 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 disruption and homogeni
39. t room temperature for 2 3 min 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 yl Transfer the upper aqueous phase to a new tube not supplied Add 1 volume usually 600 pl of 70 ethanol and mix thoroughly by pipetting up and down Do not centrifuge Proceed immediately to step 9 Note The volume of lysate may be less than 600 pl 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 9 RNeasy Microarray Tissue Handbook 11 2009 9 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 gt 8000 x g 210 000 rpm at room temperature 15 25 C Discard the flow through Reuse the collection tube in step 10 JO20 01q 10 Repeat step 9 using the remainder of the sample Discard the flow through Reuse
40. terial 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 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 4 mm cube 64 mm of most animal tissues weighs 70 85 mg Important points before starting E f using the RNeasy Microarray Tissue Mini Kit for the first time read Important Notes page 11 E f working with RNA for the first time read Appendix A page 25 If 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 E 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 QIAzol 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 QIAzol and RNeasy technologies efficiently remove most of the DNA without
41. the collection tube in step 11 Optional If performing optional on column DNase digestion see Important points before starting follow steps C1 C4 page 30 after performing this step 11 Add 700 pl Buffer RW1 to the RNeasy spin column Close the lid gently and centrifuge for 15 s at gt 8000 x g 210 000 rpm to wash the membrane Discard the flow through Reuse the collection tube in step 12 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 Skip this step if performing optional on column DNase digestion page 30 12 Add 500 pl Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge for 15 s at gt 8000 x g 210 000 rpm to wash the membrane Discard the flow through Reuse the collection tube in step 13 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 16 13 Add 500 pl Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge for 2 min at gt 8000 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 tub
42. ure 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 Perform optional on column DNase digestion using the RNase Free DNase Set Appendix C page 30 at step 10 of the protocol RNA does not perform well in downstream experiments a Salt carryover during elution b Ethanol carryover 24 Ensure that Buffer RPE is at 20 30 C During the second wash with Buffer RPE step 13 be sure to dry the RNeasy spin column membrane by centrifuging at gt 8000 x g 210 000 rpm for 2 min at 15 25 C 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 place the RNeasy Mini spin column in a new 2 ml collection tube and perform the optional 1 min centrifugation step as described in step 14 of the protocol RNeasy Microarray Tissue Handbook 11 2009 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 inadvertent
43. water see flowchart page 9 For microarray analysis of cultured cells we recommend purifying RNA using either the RNeasy Microarray Tissue Mini Kit or the RNeasy Mini Kit cat no 74104 t To ensure optimal RNA yields the binding capacity of the RNeasy spin column must not be exceeded For details see the protocol page 15 RNeasy Microarray Tissue Handbook 11 2009 7 With the RNeasy Microarray Tissue Mini Kit 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 8S 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 gradient or cushion where small RNAs do not sediment efficiently For purification of small RNA including microRNA from tissues and cells we recommend using miRNeasy Kits see ordering information page 34 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 Microarray Tissue Mini Kit for purification of
44. zation than conical bottomed tubes 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 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 Place the tubes in the insert of the TissueLyser LT Adapter and incubate at room temperature for 2 min Then immediately add 1 ml QIAzol Lysis Reagent per tube Do not incubate for longer than 2 min otherwise frozen tissues will thaw resulting in potential RNA degradation Place the tubes in the TissueLyser LT Adapter Operate the TissueLyser LT for 2 5 min at 50 Hz The time depends on the tissue being processed and can be extended until the tissue is completely homogenized Carefully
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