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RNeasy® Lipid Tissue Handbook

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1. the phases are not well separated shake the tube vigorously for at least 15 s and repeat the incubation and centrifugation in steps and 7 c Organic solvents in samples Make sure that the starting sample does not used for RNA purification contain organic solvents e g ethanol DMSO 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 homogenization material page 12 for details on disruption and homogenization methods Increase g force and centrifugation time if necessary In subsequent preparations reduce the amount of starting material see page 10 and protocol page 14 or 19 and or increase the homogenization time b Too much starting material In subsequent preparations reduce the amount of starting material It is essential to use the correct amount of starting material see page 10 and protocol page 14 or 19 24 RNeasy Lipid Tissue Handbook 02 2009 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 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 Th
2. 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 A 4 A provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV spectrum such as protein However the Ay4o Azgo ratio is influenced considerably by pH Since water is not buffered the pH and the resulting Az6o Azgo ratio can vary greatly Lower pH results in a lower Ajo A so 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 A Asso 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 for RNA at neutral pH see Spectrophotometric quantification of RNA page 30 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 For analysis of very low abundance targets any interference by r
3. 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 column Maximum binding capacity 100 pg RNA 1 mg RNA Maximum loading volume 700 yl 4 ml RNA size distribution RNA gt 200 RNA gt 200 nucleotides nucleotides Minimum elution volume 30 pl 150 pl Maximum amount of starting tissue lt 100 mg lt 500 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 10 RNeasy Lipid Tissue Handbook 02 2009 Table 2 Typical yields of total RNA wi
4. 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 o 2 N RS bud t Z E r 3 E 7 cc Skip this step if performing optional on column DNase digestion page 33 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 gt 10 000 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 15 13 Add 500 pl 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 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 Buffe
5. 1500 Kunitz units Mix gently by inverting the vial Do not vortex E For long term storage of DNase stock solution 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 aliquots after thawing Procedure Perform steps 1 7 of Bench Protocol RNA Purification Using the RNeasy Lipid Tis sue Mini Kit Then perform steps 1 4 below 1 Add 350 pl Buffer RW1 to RNeasy column close lid centrifuge for 15 s at gt 8000 x g and discard flow through 2 Add 10 pl DNase I stock solution see above to 70 pl Buffer RDD Mix by gen tly inverting tube and centrifuge briefly 3 Add DNase incubation mix 80 pl directly to RNeasy column membrane and place on benchtop 20 30 C for 15 min 4 Add 350 pl Buffer RW1 to RNeasy column close lid centrifuge for 15 s at 28000 x g and discard flow through Continue with step 9 of Bench Protocol RNA Purification Using the RNeasy Lipid Tissue Mini Kit QIAGEN BenchProtocol RNA Purification Using RNeasy Lipid Tissue Midi Kit Note Before using this bench protocol you should be completely familiar with the safety information and protocols in the RNeasy Lipid Tissue Handbook Please tear here Procedure 1 Disrupt and homogenize lt 500 mg fatty tissue lt 250 mg other tissue in 5 ml GlAzol Lysis Reagent using the TissueRuptor Incubate sample at room temperature for 5 min Add
6. a working solution If performing optional on column DNase digestion prepare DNase stock solution as described in Appendix C page 33 Procedure 1 20 Excise the tissue sample from the animal or remove it from storage Determine the amount of tissue Do not use more than 500 mg 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 Place the tissue in a suitably sized vessel containing 5 ml GlAzol Lysis Reagent Note Use a suitably sized vessel with sufficient extra headspace to accommodate foaming which may occur during homogenization RNeasy Lipid Tissue Handbook 02 2009 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 Note To avoid damage to the TissueRuptor and disposable pr
7. aqueous phase should be approximately 600 yl 8 Transfer the upper aqueous phase to a new tube not supplied Add 1 volume usually 600 pl of 70 ethanol and mix thoroughly by vortexing Do not centrifuge Proceed immediately to step 9 Note The volume of lysate may be less than 600 yl 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 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 28000 x g 210 000 rpm at room temperature 15 25 C Discard the flow through Reuse the collection tube in step 10 10 Repeat step 9 using the remainder of the sample Discard the flow through Reuse the collection tube in step 11 Optional If performing optional on column DNase digestion see Important points before starting follow steps C1 C4A page 33 after performing this step Flow through contains GlAzol Lysis Reagent and is therefore not compatible with bleach See page 6 for safety information RNeasy Lipid Tissue Handbook 02 2009 17 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 gt 10 000 rpm to wash the membrane Discard the flow through Reuse the collection tube in step 12 After centrifugation
8. bath until completely thawed and salts are dissolved before continuing with step 4 Avoid prolonged incubation which may compromise RNA integrity RNeasy Lipid Tissue Handbook 02 2009 19 E wn den RS a 5 2 5 Z eA T 3 Generally DNase digestion is not required since integrated QIAzol and RNeasy technologies efficiently remove most of the DNA without DNase treatment However further DNA removal may be necessary for certain RNA applications that are sensitive to very small amounts of DNA e g TaqMan real time RT PCR analysis with a low abundance target In these cases residual DNA can be removed by optional on column DNase digestion using the RNase Free DNase Set see Appendix C page 33 Alternatively for realtime two step RT PCR applications the QuantiTect Reverse Transcription Kit provides cDNA synthesis with integrated removal of genomic DNA contamination see ordering information page 37 GlAzol 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 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 Things to do before starting Buffer RPE is supplied as a concentrate Before using for the first time add A volumes of ethanol 96 100 as indicated on the bottle to obtain
9. be extended until the tissue is completely homogenized m Disassemble the adapter set rotate the rack of tubes so that the tubes nearest to the Tissuelyser are now outermost and reassemble the adapter set Operate the TissueLyser 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 16 RNeasy Lipid Tissue Handbook 02 2009 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 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 6 Place the tube containing the homogenate on the benchtop at room temperature for 2 3 min 7 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 z Z 9 Q wn lt zc Tt a wn c 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
10. diethyl pyrocarbonate Fill glassware with 0 1 DEPC 0 1 in water allow to stand overnight 12 hours at 37 C and then autoclave or heat to 100 C for 15 minutes to eliminate residual DEPC Electrophoresis tanks Electrophoresis tanks should be cleaned with detergent solution e g 0 5 SDS thoroughly rinsed with RNase free water and then rinsed with ethanol and allowed to dry Solutions Solutions water and other solutions should be treated with 0 1 DEPC DEPC is a strong but not absolute inhibitor of RNases It is commonly used at a concentration of 0 1 to inactivate RNases on glass or plasticware or to create RNase free solutions and water DEPC inactivates RNases by covalent modification Add 0 1 ml DEPC to 100 ml of the solution to be treated and shake vigorously to bring the DEPC into solution Let the solution incubate for 12 hours at 37 C Autoclave for 15 minutes to remove any trace of DEPC DEPC will react with primary amines and cannot be used directly to treat Tris buffers DEPC is highly unstable in the presence of Tris buffers and decomposes rapidly into ethanol and CO When preparing Tris buffers treat water with DEPC first and then dissolve Tris to make the appropriate buffer Trace amounts of DEPC will modify purine residues in RNA by carbethoxylation Carbethoxylated RNA is translated with very low efficiency in cell free systems However its ability to form DNA RNA or RNA RNA hybrids is not seriously affec
11. enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and or its components For updated license terms see www qiagen com 2002 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 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 0086 2 1 3865 3865 Fax 0086 21 3865 3965 Technical 800 988 0325 800 988 0327 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
12. 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 C4 Add A 350 pl or 2 ml Buffer RW1 to the RNeasy spin column Close the lid gently and centrifuge for A 15 s at 28000 x g 210 000 rpm or 6 5 min at 3000 5000 x g Discard the flow through Continue with step 12 of the protocol on page 14 or 19 Flow through contains Buffer RW1 and is therefore not compatible with bleach See page 6 for safety information 34 RNeasy Lipid Tissue Handbook 02 2009 Ordering Information Product Contents Cat no RNeasy Lipid Tissue 50 RNeasy Mini Spin Columns 74804 Mini Kit 50 Collection Tubes QIAzol Lysis Reagent RNase Free Reagents and Buffers RNeasy Lipid Tissue 10 RNeasy Midi Spin Columns 75842 Midi Kit 10 Collection Tubes QIAzol Lysis Reagent RNase Free Reagents and Buffers Accessories Allprotect Tissue Reagent For stabilization of DNA RNA and 76405 100 ml protein in 50 x 200 mg tissue samples 100 ml Allprotect Tissue Reagent Allprotect Reagent Pump RNAlater RNA For stabilization of RNA in 76104 Stabilization Reagent 50 ml 25 x 200 mg tissue samples 50 ml RNAlater RNA Stabilization Reagent RNAlater RNA For stabilization of RNA in 76106 Stabilization Reagent 250 ml 125 x 200 mg ti
13. 1 5 ml and 2 ml GlAzol Lysis Reagent RNase Free Reagents and Buffers miRNeasy 96 Kit 4 For 4 x 96 preps 4 RNeasy 217061 96 plates Collection Microtubes racked Elution Microtubes CL Caps S Blocks AirPore Tape Sheets QIAzol Lysis Reagent RNase Free Reagents and Buffers Allprotect Tissue Reagent RNAlater RNA Stabilization Reagent the TissueRuptor the Tissuelyser Il and RNeasy Kits are intended for molecular biology applications These products are neither intended for the diagnosis prevention or treatment of a disease nor have they been validated for such use either alone or in combination with other products Visit www qiagen com geneXpression to find out more about standardized solutions for gene expression analysis from RNA preparation to real time RT PCR t Larger kit size available see www giagen com RNA Requires use of the QIAGEN 96 Well Plate Centrifugation System with refrigeration capability Tissuelyser system recommended for disruption and homogenization QlAvac 96 optional 38 RNeasy Lipid Tissue Handbook 02 2009 Trademarks QIAGEN QIAxcel QIAzol Quantiscript QuantiTect RNeasy TissueRuptor QIAGEN Group Agilent Agilent Technologies Inc Applied Biosystems Applera Corporation or its subsidiaries Rotor Gene Corbett Research SYBR Molecular Probes Inc TaqMan Roche Group RNAlater is a trademark of AMBION Inc Austin Texas and is covered b
14. 1 ml chloroform and shake vigorously for 15 s Incubate sample at room temperature for 2 3 min Centrifuge at 5000 x g for 15 min at 4 C Transfer upper aqueous phase to new tube add 1 volume of 70 ethanol and vortex Do not centrifuge Proceed at once to step 7 Soa PWN 7 Transfer sample to RNeasy column in 15 ml tube Close lid centrifuge for 5 min at 3000 5000 x g and discard flow through Optional DNase digest Follow steps 1 4 of Bench Protocol Optional On Column DNase Digestion for RNeasy Lipid Tissue Midi Kit after this step 8 Add 4 ml Buffer RW1 to RNeasy column Close lid centrifuge for 5 min at 3000 5000 x g and discard flow through Skip this step if performing optional DNase digestion 9 Add 2 5 ml Buffer RPE to RNeasy column Close lid centrifuge for 2 min at 3000 5000 x g and discard flow through 10 Add 2 5 ml Buffer RPE to RNeasy column Close lid and centrifuge for 5 min at 3000 5000 x g 11 Place RNeasy column in new 15 ml tube Add RNase free water 150 pl if expecting lt 150 pg RNA or 250 pl if expecting lt 1 mg RNA close lid wait 1 min and centrifuge for 3 min at 3000 5000 x g Optional Repeat elution with another volume of water or with RNA eluate QIAGEN BenchProtocol Optional On Column DNase Digestion for RNeasy Lipid Tissue Midi Kit Note Before using this bench protocol you should be completely familiar with the safety information and detailed protocols in the RNea
15. February 2009 RNeasy Lipid Tissue Handbook RNeasy Lipid Tissue Mini Kit RNeasy Lipid Tissue Midi Kit For purification of total RNA from fatty tissues and all other types of tissue 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 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 qgiagen com Contents Kit Contents 4 Storage 4 Quality Control 4 Product Use Limitations 5 Product Warranty and Satisfaction Guarantee 5 Technical Assistance 5 Safety Information 6 Introduction 7 Principle and procedure 7 Equipment and Reagents to Be Supplied by User 9 Important Notes 10 Determining the amount of starting material 10 Handling and storing starting material 11 Disrupting and homogenizing starting material 12 Protocols E Purification of Total RNA Using the RNeasy Lipid Tissue Mini Kit 14 H Purification of Total RNA Using the RNeasy Lipid Tissue Midi Kit 19 Troubleshooting Guide 24 Appendix A General Remarks on Handling RNA 28 Appendix B Storage Quantification
16. La rs oo jg E 5 7 4 Reuse the collection tube in step 12 Skip this step if performing optional on column DNase digestion page 33 12 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 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 20 13 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 14 Place the RNeasy spin column in a new 15 ml collection tube supplied Add the appropriate volume of RNase free water see Table 3 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 Flow through contains GlAzol Lysis Reagent or Buffer RW1 and is therefore not compatible with bleach See page 6 for safety information 22 RNeasy Lipid Tissue Handboo
17. Mexico Orders 01 800 7742 639 Fax 01 800 1122 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 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 Qe e e UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 e o o e e USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN T Sample amp Assay Technologies BenchProtocol RNA Purification Using RNeasy Lipid Tissue Mini Kit Note Before using this bench protocol you should be completely familiar with the safety information and protocols in the RNeasy Lipid Tissue Handbook Please tear here Procedure 1 Disrupt and homogenize lt 100 mg fatty tissue lt 50 mg other tissue in 1 ml GlAzol Lysis Reagent using the TissueRuptor or TissueLyser Incubate sample at room temperature for 5 min Add 200 pl chloroform and shake vigorously for 15 s Incubate sample at room temperature for 2 3 min Centrifuge at 12 000 x g for 15 min at 4 C Transfer upper aqueous phase to new tube add 1 volume of 70 ethanol and vortex Do not centrifuge Proceed at once to
18. and Determination of Quality of RNA 30 Appendix C Optional On Column DNase Digestion with the RNase Free DNase Set 33 Ordering Information 35 RNeasy Lipid Tissue Handbook 02 2009 3 Kit Contents RNeasy Lipid Tissue Kit Mini 50 Midi 10 Catalog no 74804 75842 Number of preps 50 10 RNeasy Mini Spin Columns each in a 2 ml Collection Tube 50 RNeasy Midi Spin Columns each in a 15 ml Collection Tube 10 Collection Tubes 1 5 ml 50 Collection Tubes 2 ml 50 Collection Tubes 15 ml 10 QIAzol Lysis Reagent 50 ml 50 ml Buffer RW 1 45 ml 45 ml Buffer RPE concentrate 11 ml 11 ml RNase Free Water 10 ml 10 ml Handbook 1 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 96 100 as indicated on the bottle to obtain a working solution Note GlAzol Lysis Reagent is delivered separately Storage RNeasy Lipid Tissue Kits should be stored dry at room temperature 15 25 C All components are stable for at least 9 months under these conditions GlAzol 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 Lipid Tissue Mini Kit and RNeasy Lipid Tissue Midi Kit is tested against predetermined specifications to ensure consistent product quality 4 RNeasy Lip
19. column Average RNA yields from various tissues are given in Table 2 page 11 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 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 df using RNeasy Lipid Tissue Kits for the first time read Important Notes page 10 Wb If working with RNA for the first time read Appendix A page 28 HM If using the TissueRuptor ensure that you are familiar with operating it by referring to the TissueRuptor User Manual and TissueRuptor Handbook MH If using the Tissuelyser ensure that you are familiar with operating it by referring to the operating instructions and Tissuelyser 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 d
20. d 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 with Buffer RW1 Washing with Buffer RPE and elution of RNA are then performed according to the standard protocol Important points before starting B Generally DNase digestion is not required since integrated QIAzol and RNeasy technologies efficiently remove most of the DNA without DNase treatment However further DNA removal may be necessary for certain RNA applications that are sensitive to very small amounts of DNA e g TaqMan RT PCR analysis with a low abundant target DNA can also be removed by a DNase digestion following RNA purification E Do not vortex the reconstituted DNase I DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the tube Bb Inthe procedure below A refers to use of the RNeasy Lipid Tissue Mini Kit and refers to use of the RNeasy Lipid Tissue Midi Kit Things to do before starting E Prepare DNase stock solution before using the RNase Free DNase Set for the first time Dissolve the lyophiliz
21. d 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 Lipid Tissue Handbook 02 2009 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 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 Perform optional on column DNase digestion using the RNase Free DNase Set Appendix C page 33 at step 10 of the protocol RNA does not perform well in downstream experiments a Salt carryover during elution b Ethanol carryover RNeasy Lipid Tissue Handbook 02 2009 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 RNeasy Lipid Tiss
22. d homogenize tissues 12 RNeasy Lipid Tissue Handbook 02 2009 The Tissuelyser can 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 RNeasy Lipid Tissue Kits For ordering information see page 38 RNeasy Lipid Tissue Handbook 02 2009 13 2 wn En 2 a S el a 5 7 Ze a Protocol Purification of Total RNA Using the RNeasy Lipid Tissue 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 Lipid 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 QIAzol 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
23. described in Appendix C page 33 Procedure 1 If using the TissueLyser 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 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 RNA ater 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 RNeasy Lipid Tissue Handbook 02 2009 15 z Z 9 Q wn lt zc Tt a wn c 2 wn En 2 a S el a 5 7 7 a 3 Disrupt the tissue and homogenize the lysate using either the TissueRupter follow step 3a or Tissuelyser follow step 3b See Disrupting and homogenizing starting material page 12 for more details on disruption and homogenization Note Incomplete homogenization leads to significantly reduced RNA yiel
24. ds and can cause clogging of the RNeasy spin column Homogenization with the TissueRupter or Tissuelyser generally results in higher RNA yields than with other methods 3a Disruption and homogenization using the TissueRuptor m Place the tissue in a suitably sized vessel containing 1 ml GlAzol 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 homogenization than conical bottomed tubes m 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 m Place the tissues in the tubes prepared in step 1 m Ifthe tubes were stored on dry ice place them at room temperature Then immediately add 1 ml QIAzol Lysis Reagent per tube m Place the tubes in the Tissuelyser Adapter Set 2 x 24 EH Operate the TissueLyser for 2 min at 20 Hz The time depends on the tissue being processed and can
25. e Free Buffer RDD and RNase Free Water 100 x 15 ml MaXtract tubes for 100 nucleic acid extractions 25 x 50 ml MaXtract tubes for 25 nucleic acid extractions 1000 x 2 ml Collection Tubes 2 sets of Adapter Plates for use with Collection Microtubes racked on the Tissuelyser Nonsterile polypropylene tubes 1 2 ml 960 in racks of 96 Nonsterile polypropylene caps for collection microtubes 1 2 ml 960 in strips of 8 120 V 60 Hz 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 36 Cat no 9001271 9001272 9001273 90012748 990890 85300 69982 69965 69989 79254 129065 129073 19201 69984 19560 19566 RNeasy Lipid Tissue Handbook 02 2009 Ordering Information Product Contents Cat no Related products QuantiTect Reverse Transcription Kit for fast cDNA synthesis for sensitive real time two step RT PCR QuantiTect Reverse For 50 x 20 yl reactions gDNA 205311 Transcription Kit 50 Wipeout Buffer Quantiscript Reverse Transcriptase Quantiscript RT Buffer RT Primer Mix and RNase Free Water RNeasy Kits for purification of total RNA from animal cells or tissues or yeast RNeasy Mini Kit 50 50 RNeasy Mini Spin Columns 74104 Collection Tubes RNase Free Reagents and Buffers RNeasy Midi Kit 10 10 RNeasy Midi Spin Columns 75142 Collection Tub
26. ed 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 l 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 RNeasy Lipid Tissue Handbook 02 2009 33 Procedure Prepare and load samples onto the RNeasy spin column as indicated in steps 1 10 of the protocol on page 14 or 19 Instead of performing step 11 follow steps C1 C4 below Cl Add A 350 pl or 2 ml Buffer RW1 to the RNeasy spin column Close the lid gently and centrifuge for A 15 s at 28000 x g 210 000 rpm or 5 min at 3000 5000 x g to wash the membrane Discard the flow through Reuse the collection tube in step C4 C2 Add A 10 pl or 6 20 pl DNase I stock solution see above to A 70 pl or 140 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 C3 Add the DNase incubation mix A 80 pl or 160 pl directly to
27. een and thymus we recommend using no more than 150 mg tissue fo 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 11 amp 5 anssiy pidi Aspany 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 E df using RNeasy Lipid Tissue Kits for the first time read Important Notes page 10 E f working with RNA for the first time read Appendix A page 28 E If using the TissueRuptor ensure that you are familiar with operating it by referring to the TissueRuptor User Manual and TissueRuptor 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 GlAzol 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
28. es RNase Free Reagents and Buffers RNeasy Plus Mini Kit for purification of total RNA from cultured cells and tissues using gDNA Eliminator columns RNeasy Plus Mini Kit 50 50 RNeasy Mini Spin Columns 74134 50 gDNA Eliminator Mini Spin Columns Collection Tubes RNase Free Reagents and Buffers RNeasy Fibrous Tissue Kits for purification of total RNA from fiber rich tissues RNeasy Fibrous Tissue 50 RNeasy Mini Spin Columns 74704 Mini Kit 50 Collection Tubes Proteinase K RNase Free DNase RNase Free Reagents and Buffers RNeasy Fibrous Tissue 10 RNeasy Midi Spin Columns 75742 Midi Kit 10 Collection Tubes Proteinase K RNase Free DNase RNase Free Reagents and Buffers Larger kit size available see www giagen com products PCR t Larger kit sizes and format available see www giagen com RNA RNeasy Lipid Tissue Handbook 02 2009 37 Ordering Information Product Contents Cat no 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 74881 Tissue Kit 4 4 RNeasy 96 Plates Collection Microtubes Elution Microtubes CL Caps S Blocks AirPore Tape Sheets GlAzol 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 217004 Columns Collection Tubes
29. esidual 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 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 qiagen com GeneGlobe For real time RT PCR assays where amplification 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 37 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 RNeasy Lipid Tissue Handbook 02 2009 31 For other sensitive applications DNase digestion of the purified RNA with RNase free DNase is recommended A protocol for optional on column DNase digestion using the RNase Free DNase Set is provided in Appendix C page 33 The DNase is efficien
30. etails see the individual protocols RNeasy Lipid Tissue Handbook 02 2009 7 RNeasy Lipid Tissue RNeasy Lipid Tissue Mini Procedure Midi Procedure Tissue Tissue fos lyse and i S homogenize lyse and homogenize Add chloroform and shake ow Add chloroform e Separate phases and shake 1 cP Separate phases Add ethanol to aqueous phase w y Bind total RNA Add ethanol et 1 to aqueous phase a Wash Bind total RNA et Elute Total RNA Wash e Elute m e Total RNA 8 RNeasy Lipid Tissue Handbook 02 2009 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 B Chloroform Ethanol 70 and 96 100 Sterile RNase free pipet tips Equipment for tissue disruption and homogenization see page 12 we recommend either the TissueRuptor with TissueRuptor Disposable Probes or the Tissuelyser system see ordering information page 36 E For stabilization of RNA in tissues see page 11 RNAlater RNA Stabilization Reagent or Allprotect Tissue Reagent see ordering information page 35 or liquid nitrogen and dry ice Users of RNeasy Lipid Tissue Mini Kit B 1 5 ml or 2 ml microcentrifuge tubes B Microcentrifuge s with rotor for 2 ml tubes for centrifugation at 4 C and at room temperature 15 25 C Users
31. f swallowed R68 Possible risk of irreversible effects 324 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 Lipid Tissue Handbook 02 2009 Introduction RNeasy Lipid Tissue Kits are designed for optimal lysis of tissues rich in fat such as brain and adipose tissue and purification of high quality total RNA The kits are also compatible with all other types of animal tissue QIAGEN 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 Lipid Tissue Kits integrate phenol guanidine based sample lysis and silica membrane purification of total RNA QIAzol Lysis Reagent included in the kits is a monophasic solution of phenol and guanidine thiocyanate designed to facilitate lysis of fatty tissues and inhibit RNases The high lysis efficiency of the reagent and the sub sequent removal of contaminants by organic phase extraction enables use of larger amounts of tissue with RNeasy spin columns HW RNeasy Lipid Tissue Mini Kit up to 100 mg of brain or adipose tissue per RNeasy Mini spin column up to 50 mg of other animal tissues E RNeasy lipid Tissue Midi K
32. formance please call QIAGEN Technical Services or your local distributor see back cover or visit www giagen 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 Lipid Tissue Kits or QIAGEN products in general please do not hesitate to contact us GIAGEN 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 GIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please see our Technical Support Center at www giagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www giagen com RNeasy Lipid Tissue Handbook 02 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 PDF format at ww
33. id Tissue Handbook 02 2009 Product Use Limitations RNeasy Lipid Tissue Kits are intended for molecular biology applications These products are neither intended for the diagnosis prevention or treatment of a disease nor have they been validated for such use either alone or in combination with other products Therefore the performance characteristics of these products for clinical use i e diagnostic prognostic therapeutic or blood banking are unknown Product Warranty and Satisfaction Guarantee GIAGEN 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 GIAGEN 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 per
34. ignificance Aygo readings should be greater than 0 15 An absorbance of 1 unit at 260 nm corresponds to 44 pg of RNA per ml Axo 1 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 31 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 29 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 absorbance of diluted sample in a 1 ml cuvette RNase free Axo 0 2 Concentration of RNA sample 44 pg ml x Azo x dilution factor 44 pg ml x 0 2 x 50 440 pg ml When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier 30 RNeasy Lipid Tissue Handbook 02 2009 Total amount
35. is 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 12 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 10 and protocol page 14 or 19 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 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 Lipid Tissue Mini Kit RNase free water incorrectly dispensed RNeasy Lipid Tissue Handbook 02 2009 Add RNase free water to the center of the RNeasy spin column membrane to ensure that the membrane is completely covered 25 Comments and suggestions Low A Asso value a Not enough QIAzol Lysis Reagent used for homogenization b Sample not incubated for 5 mi
36. issolved before continuing with step 4 Avoid prolonged incubation which may compromise RNA integrity 14 RNeasy Lipid Tissue Handbook 02 2009 Generally DNase digestion is not required since integrated QIAzol and RNeasy technologies efficiently remove most of the DNA without DNase treatment However further DNA removal may be necessary for certain RNA applications that are sensitive to very small amounts of DNA e g TaqMan real time RT PCR analysis with a low abundance target In these cases residual DNA can be removed by optional on column DNase digestion using the RNase Free DNase Set see Appendix C page 33 Alternatively for realtime two step RT PCR applications the QuantiTect Reverse Transcription Kit provides cDNA synthesis with integrated removal of genomic DNA contamination see ordering information page 37 GlAzol 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 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 Things to do before starting 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 If performing optional on column DNase digestion prepare DNase stock solution as
37. it up to 500 mg of brain or adipose tissue per RNeasy Midi spin column up to 250 mg of other animal tissues Tissue samples are homogenized in GlAzol 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 water see flowcharts next page With RNeasy Lipid Tissue Kits 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 38 To ensure optimal RNA yields the binding capacity of the RNeasy spin column must not be exceeded For d
38. k 02 2009 Table 3 Volumes of RNase Free Water for RNA Elution RNase free water 150 pl 250 yl Expected total RNA yield lt 150 pg 150 pg 1 mg 15 Repeat step 14 using another volume of RNase free water or using the eluate from step 14 if high RNA concentration is required Reuse the collection tube from step 14 If using the eluate from step 14 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 wa z 1 e 7 x lt Te a E 7 nn 7 RNeasy Lipid Tissue Handbook 02 2009 23 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 giagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www giagen com Comments and suggestions Phases do not separate completely a No chloroform added or Make sure to add chloroform that does not chloroform not pure contain isoamyl alcohol or other additives b Homogenate not sufficiently After addition of chloroform step 5 the mixed before centrifugation homogenate must be vigorously shaken If
39. n 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 30 For frozen tissue samples ensure that they were 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 28 and Handling and storing starting material page 11 Although all RNeasy buffers have been tested and are guaranteed RNase ree RNases can be introduced during use Be certain not to introduce any RNases during the RNeasy procedure or later handling See Appendix A page 28 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 26 The phase separation step 7 should be performe
40. obe 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 amp 5 nss pidi Aspany 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 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 6 Place the tube containing the homogenate on the benchtop at room temperature for 2 3 min 7 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 Optional Alternatively centrifuge the h
41. of RNeasy Lipid Tissue Midi Kit B 10 15 ml centrifuge tubes HM Laboratory centrifuge s capable of 5000 x g for centrifugation at 4 C and at room temperature 15 25 C E Optional MaXtract High Density see ordering information page 36 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 RNeasy Midi spin columns can also be inserted into different 12 15 ml RNase free glass or polypropylene tubes RNeasy Lipid Tissue Handbook 02 2009 9 Important Notes Determining the amount of starting material It is essential fo 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 BW The volume of QIAzol Lysis Reagent required for efficient lysis B The RNA binding capacity of the RNeasy spin column When processing samples containing high amounts of RNA less than the maximum
42. omogenate in a 15 ml or 50 ml MaXtract High Density tube for ordering information see page 36 The MaXtract tube contains a gel which forms a stable barrier between the aqueous phase and the other phases after centrifugation 8 Transfer the upper aqueous phase to a new tube not supplied Add 1 volume usually 3 ml of 70 ethanol and mix thoroughly by vortexing Do not centrifuge Proceed immediately to step 9 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 9 RNeasy Lipid Tissue Handbook 02 2009 21 9 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 10 10 Repeat step 10 using the remainder of the sample up to 4 ml Discard the flow through Reuse the collection tube in step 11 Optional If performing optional on column DNase digestion see Important points before starting follow steps C1 C4 page 33 after performing this step 11 Add 4 ml Buffer RW1 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 oO D N a
43. r RPE or if residual flow through remains on the outside of the RNeasy spin column after step 13 15 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 28000 x g 210 000 rpm 16 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 Flow through contains Buffer RW1 and is therefore not compatible with bleach See page 6 for safety information 18 RNeasy Lipid Tissue Handbook 02 2009 Protocol Purification of Total RNA Using the RNeasy Lipid Tissue 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 Lipid Tissue Midi Kit a maximum of 500 mg brain or adipose tissue can generally be processed For these tissues the RNA binding capacity of the RNeasy Midi spin column and the lysing capacity of QIAzol Lysis Reagent will not be exceeded by these amounts For other tissues a maximum of 250 mg tissue can generally be used For tissues with high RNA content such as liver spl
44. ssue 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 RNA ater 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 GlAzol Lysis Reagent 200 ml 200 ml GlAzol Lysis Reagent 79306 RNeasy Lipid Tissue Handbook 02 2009 35 Ordering Information Product TissueRuptor TissueRuptor Disposable Probes 25 Tissuelyser II Tissuelyser Adapter Set 2 x 24 Tissuelyser Single Bead Dispenser 5 mm Stainless Steel Beads 5 mm 200 RNase Free DNase Set 50 MaxXtract High Density 100 x 15 ml MaxXtract High Density 25 x 50 ml Collection Tubes 2 ml Tissuelyser Adapter Set 2x96 Collection Microtubes racked Collection Microtube Caps 120 x 8 Contents Handheld rotor stator homogenizer 5 TissueRuptor Disposable Probes 25 nonsterile plastic disposable probes for use with the TissueRuptor Universal laboratory mixer mill disruptor 2 sets of Adapter Plates and 2 racks for use with 2 ml microcentrifuge tubes on the Tissuelyser For dispensing individual beads 5 mm diameter Stainless Steel Beads suitable for use with the Tissuelyser system For 50 RNA minipreps 1500 units RNase Free DNase I RNas
45. step 7 Soa PWN 7 Transfer sample to RNeasy column in 2 ml tube Close lid centrifuge for 15 s at 28000 x g and discard flow through Optional DNase digest Follow steps 1 4 of Bench Protocol Optional On Column DNase Digestion for RNeasy Lipid Tissue Mini Kit after this step 8 Add 700 pl Buffer RW1 to RNeasy column Close lid centrifuge for 15 s at 28000 x g and discard flow through Skip this step if performing optional DNase digestion 9 Add 500 yl Buffer RPE to RNeasy column Close lid centrifuge for 15 s at 28000 x g and discard flow through 10 Add 500 pl Buffer RPE to RNeasy column Close lid and centrifuge for 2 min at 28000 x g 11 Optional Place RNeasy column in new 2 ml tube close lid and centrifuge at full speed for 1 min 12 Place RNeasy column in new 1 5 ml tube Add 30 50 pl RNase free water close lid and centrifuge for 1 min at 28000 x g Optional Repeat elution with another volume of water or with RNA eluate QIAGEN BenchProtocol Optional On Column DNase Digestion for RNeasy Lipid Tissue Mini Kit Note Before using this bench protocol you should be completely familiar with the safety information and detailed protocols in the RNeasy Lipid Tissue Handbook Please tear here Things to do before starting BE f using the RNase Free DNase Set for the first time prepare DNase stock solution Using an RNase free needle and syringe inject 550 pl RNase free water into the DNase vial
46. sy Lipid Tissue Handbook Please tear here Things to do before starting BE f using the RNase Free DNase Set for the first time prepare DNase stock solution Using an RNase free needle and syringe inject 550 pl RNase free water into the DNase vial 1500 Kunitz units Mix gently by inverting the vial Do not vortex E For long term storage of DNase stock solution 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 aliquots after thawing Procedure Perform steps 1 7 of Bench Protocol RNA Purification Using the RNeasy Lipid Tis sue Midi Kit Then perform steps 1 4 below 1 Add 2 ml Buffer RW1 to RNeasy column close lid centrifuge for 5 min at 3000 5000 x g and discard flow through 2 Add 20 pl DNase I stock solution see above to 140 pl Buffer RDD Mix by gently inverting tube and centrifuge briefly 3 Add DNase incubation mix 160 pl directly to RNeasy column membrane and place on benchtop 20 30 C for 15 min 4 Add 2 ml Buffer RW1 to RNeasy column close lid centrifuge for 5 min at 3000 5000 x g and discard flow through Continue with step 9 of Bench Protocol RNA Purification Using the RNeasy Lipid Tissue Midi Kit QIAGEN
47. ted 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 t Plastics used for some electrophoresis tanks are not resistant to ethanol Take proper care and check the supplier s instructions RNeasy Lipid Tissue Handbook 02 2009 29 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 A 4 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 s
48. th RNeasy Lipid Tissue Kits Mouse rat tissue 10 mg Yield of total RNA pg 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 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 RNAlafer 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 b
49. tly washed away in subsequent wash steps Integrity of RNA The integrity and size distribution of total RNA purified with RNeasy Lipid Tissue Kits can be checked by denaturing agarose gel electrophoresis and ethidium bromide staining or by using the GlAxcel system or Agilent 2100 bioanalyzer The respective ribosomal RNAs should appear as sharp bands or peaks The apparent ratio of 28S rRNA to 185 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 how well the original sample is preserved greotly influences RNA quality 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 Lipid Tissue Handbook 02 2009 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 Standar
50. ue Mini Kit or at 3000 5000 x g for 5 min at 15 25 C RNeasy Lipid Tissue Midi Kit 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 Lipid Tissue Mini Kit 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 27 Appendix A General Remarks on Handling RNA Handling RNA Ribonucleases RNases are very stable and active enzymes that generally do not require cofactors to function Since RNases are difficult to inactivate and even minute amounts are sufficient to destroy RNA do not use any plasticware or glassware without first eliminating possible RNase contamination Great care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure In order to create and maintain an RNase free environment the following precautions must be taken during pretreatment and use of disposable and nondisposable vessels and solutions while working with RNA General handling Proper microbiological aseptic technique should always be used when working with RNA Hands and dust particles may carry bacteria and molds and are the most common sources of RNase contamination Always wear latex or vinyl gloves
51. ut 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 RNeasy Lipid Tissue Handbook 02 2009 11 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 MH 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 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 the Tissuelyser for processing multiple samples simultaneously Disruption and homogenization
52. w 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 GlAzol 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 RNeasy Lipid Tissue Kits GlAzol 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 RIO 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 inhalation in contact with skin and i
53. 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 Disposable plasticware The use of sterile disposable polypropylene tubes is recommended throughout the procedure These tubes are generally RNase free and do not require pretreatment to inactivate RNases Nondisposable plasticware Nondisposable plasticware should be treated before use to ensure that it is RNase free Plasticware should be thoroughly rinsed with 0 1 M NaOH 1 mM EDTA followed by RNase free water see Solutions page 29 Alternatively chloroform resistant plasticware can be rinsed with chloroform to inactivate RNases When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier 28 RNeasy Lipid Tissue Handbook 02 2009 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
54. with the TissueRuptor or Tissuelyser 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 the TissueLyser 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 The Tissuelyser 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 refer to the Tissuelyser Handbook For other bead mills refer to suppliers guidelines Note Tungsten carbide beads react with QIAzol Lysis Reagent and must not be used to disrupt an
55. y various U S and foreign patents GlAzol 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 Lipid Tissue Mini Kit or RNeasy Lipid Tissue Midi Kit to the following terms i The RNeasy Lipid Tissue Mini Kit or RNeasy Lipid Tissue Midi Kit may be used solely in accordance with the RNeasy Lipid Tissue 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 Lipid Tissue Handbook and additional protocols available at www qiagen 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

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