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RNeasy 96 BioRobot 8000
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1. 84 4 51 19452 Ecuador Latvia Abdulla Fouad Holding Company Fax 84 4 51 19453 INMUNOCHEM S A C SIA JLM Tel 03 8324400 Email VietanhHNGhn vnn vn Tel 51 1 4409678 Tel 7136393 Fax 03 8346174 Fax 51 1 4223701 Fax 7136394 Email All other countries E mail inmunochem terra com pe Email jim amp mednet lv sadiq omar abdulla fouad com QIAGEN GmbH Germany RNeasy 96 BioRobot 8000 Handbook 10 2006 39 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 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 021 51345678 Fax 021 51342500 Technical 021 51345678 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 02 103 29 12400 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 02 3343041 1 Fax 02 33430426 Technical 800 787980 Japan Telephone 03 5547 0811 Fax 03 5547 0818 Technical 03 5547 081 1 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 The Netherlands Orders 0800 0229592 Fax 08
2. 35 ml Buffer RW1 Reagent carousel Rotor Slot 2 165 ml 271 ml Buffer RPE Reagent carousel Rotor Slot 4 251 ml 443 ml Ethanol Reagent carousel Rotor Slot 6 145 ml 190 ml 96 100 Distilled Reagent carousel Rotor Slot 8 700 ml 700 ml water RNase free Reagent holder for 8 x 2 ml tubes water MP Slot 8 Reagent Holder Tray B 8 x 1 9 ml 8 x 1 9 ml Reagent holder for 8 x 2 ml tubes MP Slot 8 Reagent Holder Tray 8 x 1 9 ml Top Elute Reagent holder for 8 x 1 5 ml tubes Fluid MP Slot 8 Reagent Holder Tray A Ax148ml 8x1 48 ml Optional Reagent holder for 8 x 1 5 ml tubes RNase free VariTherm Slot Reagent DNase It X Holder Tray A 4x 1 9 ml 8 x 1 9 ml Before using Buffer RPE for the first time be sure to add 4 volumes of ethanol 96 100 t See Appendix D page 30 for details on preparing RNase free DNase 14 RNeasy 96 BioRobot 8000 Handbook 10 2006 Table 4 Loading Plasticware BioRobot Universal System Item Position Holder adapter RNeasy 96 plate QlAplate Holder silver 11 Silver multiwell plate holder 96 well cell High speed shaker system culture plate Shaker back left S Block High speed shaker system Shaker frontleft Elution MP Slot 21 Blue elution microtube Microtubes CL adapter Channeling block QlAplate Holder black 16 Black multiwell plate holder Rack of Tip Rack Slot 2 3 4 5 7 Red tip tray holders disposable fille 10 12 13 14 20 25 tips 1100 pl Addition
3. 8000 Handbook 10 2006 Important Notes Amount of cells The recommended amount of starting material is up to 5 x 10 animal or human cells Direct counting is the most accurate way to quantify the number of cells A 96 well cell culture plate with a growth area of 0 32 0 6 cm per well depending on the supplier typically contains 4 5 x 10 confluent Hela cells per well Table 1 gives specifications for the RNeasy 96 plate Each well of the plate can bind up to 100 pg RNA but the amount of RNA in up to 5 x 105 cells is significantly less than this binding capacity Expected RNA yields are therefore less than 100 pg RNA and vary depending on the sample Table 2 shows expected RNA yields from various cell types Table 1 RNeasy 96 Plate Specifications Preps per plate 96 Amount of starting material Up to 5 x 10 cells Maximum binding capacity per well 100 pg RNA Maximum loading volume per well 1 ml RNA size distribution RNA gt 200 nucleotides Table 2 Typical Total RNA Yields with the RNeasy 96 BioRobot 8000 Kit RNA yield Cell line Source per 10 cells Hela Human cervical carcinoma 1 6 LMH Chicken hepatoma 1 3 COS 7 Monkey kidney SV 40 transformed 3 1 Huh7 Human hepatoma 2 Jurkat Human T cell leukemia 1 4 K 562 Human chronic myelogenous leukemia in blast crisis 1 9 Amounts vary due fo factors such as species developmental stage and growth conditions Since RNeasy procedure enriches
4. Total amount concentration x volume of sample in milliliters 88 pg ml x 0 1 ml 8 8 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 such as protein However the ratio is influenced considerably by pH Since water is not buffered the pH and the resulting ratio can vary greatly Lower pH results in a lower A 4o Asso 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 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 26 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 To prevent any interference by DNA in RT PCR applications such as Applied Biosystems and LightCycler RT PCR analyses we recommend designing primers that anneal at intron splice junc
5. are performed sequentially in 2 separate reactions This can be carried out using the QuantiTect Reverse Transcription Kit for reverse transcription followed by PCR using a QuantiTect PCR Kit For one step RT PCR analysis using a QuantiTect RT PCR Kit both reactions are performed in the same tube on a real time cycler Generally one step RT PCR is more commonly used Some guidelines for setting up quantitative real time RT PCR analysis and determining the linear range of the system are given below 1 Purify RNA from cells using the RNeasy 96 BioRobot 8000 Kit For the elution step elute with 80 140 pl RNase free water 2 For quantitative results the amount of input RNA must be within the linear response range of the realtime assay which may vary with the primers used and the transcripts assayed In order to determine the optimal linear range of input RNA for a specific system run a series of trial assays with 1 2 4 6 8 and 10 pl of an RNeasy 96 eluate in a 25 pl reaction volume For statistical significance we recommend assaying each volume in triplicate and repeating the triplicated series of assays at least once 3 Plot the resulting threshold cycle against the logarithm of the eluate volume Figure 2 shows an example of such an experiment with a linear response over the entire range Note that for some systems the linear response will not cover the full range Volumes outside the linear range will not yield quantitative resu
6. cycles when PCR efficiency is at its highest provides precise data for accurate quantification High Quality RNA for Sensitive Analysis of a Low Copy Transcript Threshold cycle N N w w A 1 10 100 1000 10000 100000 Cell number Figure 1 RNA was purified from 1 to 1 x 10 Hela cells using the RNeasy 96 BioRobot 8000 procedure Total RNA was eluted in 100 yl RNase free water and 5 pl was used for RT PCR Quantitative real time one step RT PCR analysis was carried out on an ABI PRISM Sequence Detection System using the QuantiTect Probe RT PCR Kit with primers and probe specific for the low copy c fos transcript EY Amplification plot Bl C values Error bars represent standard deviation from 8 different samples for each cell number 32 RNeasy 96 BioRobot 8000 Handbook 10 2006 For transcription analysis and quantification quantitative RT PCR assays require the highest quality RNA TaqMan technology was used in the development and evaluation of RNeasy 96 Kits and RNA purified with RNeasy 96 Kits continues to be thoroughly tested by TaqMan and other real time analyses RNeasy 96 Kits are the only high throughput total cellular RNA purification system providing RNA that meets stringent TaqMan standards Guidelines for quantitative RT PCR analysis Quantitative real time RT PCR analysis can be carried out in a two step or one step format In two step RT PCR analysis reverse transcription and PCR quantification
7. for details on preparing RNase free DNase 16 RNeasy 96 BioRobot 8000 Handbook 10 2006 Table 6 Loading Plasticware BioRobot Gene Expression Real Time RT PCR or BioRobot 8000 Position Holder adapter RNeasy 96 plate 96 well cell culture plate S Block Elution Microtubes CL Channeling block Rack of disposable filter tips 1100 pl RNeasy 96 plate 96 well cell culture plate Elution Microtubes CL QlAplate Slot 6 High speed shaker system Shaker back left High speed shaker system Shaker frontleft MP Slot 21 GlAplate Slot 16 Tip Rack Slot 3 4 5 8 9 10 20 QlAplate Slot 7 High speed shaker system Shaker back right MP Slot 26 QIAGEN multiwell plate Holder Blue elution microtube adapter Black multiwell plate holder Red tip tray holders Additional plasticware if processing 192 samples in one run QIAGEN multiwell plate holder Blue elution microtube adapter RNeasy 96 BioRobot 8000 Handbook 10 2006 o 2 p 2 Protocol Purification of Total RNA from Animal or Human Cells Important points before starting If preparing RNA for the first time read Appendix A page 24 If using the RNeasy 96 BioRobot 8000 Kit for the first time read Important Notes page 11 Generally DNase digestion is not required since RNeasy 96 technology efficiently removes most of the DNA without DNase treatment However furthe
8. fully automated high throughput applications in systems biology in 96 well format BioRobot Universal System Robotic workstation computer 9001094 controlled vacuum pump computer GlAsoft 5 Operating System installation 1 year warranty on parts and labor Application Pack Gene Protocols and application specific 9016754 Expression accessories for RNA purification and RT PCR setup BioRobot 8000 for flexible automation for purification of DNA or RNA reaction setup and reaction cleanup in 96 well format BioRobot 8000 Robotic workstation with selected 900500 system components computer GlAsoft 4 2 Operating System installation training and 1 year warranty on parts and labor Accessories Disposable Filter Tips Conducting disposable filter tips 9012598 1100 pl 960 pack of 960 S Blocks 24 96 well blocks with 2 2 ml wells 19585 24 per case Warranty PLUS 2 cat no 9239573 recommended 3 year warranty 1 preventive maintenance visit per year 48 hour priority response all labor travel and parts t Warranty PLUS 2 cat no 9236465 recommended 3 year warranty 1 preventive maintenance visit per year 48 hour priority response all labor travel and parts RNeasy 96 BioRobot 8000 Handbook 10 2006 35 Ordering Information Product Contents Cat no Elution Microtubes CL Nonsterile polypropylene tubes 19588 24 x 96 0 85 ml maximum capacity less than 0 7 ml storage capacity 0 4 ml elution capacity 2304 i
9. mix per tube and keep on ice until ready to load on the BioRobot workstation Note Use of other tubes may require modification of the QIAsoft protocol for assistance contact QIAGEN Other tubes also may not fit in the reagent holder 8 tube 1 5 ml cat no 9011758 Appendix E RT PCR and Real Time RT PCR RT PCR To perform PCR using RNA as a starting template the RNA must first be reverse transcribed into cDNA in a reverse transcription RT reaction RT and PCR can be carried out either sequentially in the same tube one step RT PCR or separately two step RT PCR One step RT PCR requires gene specific primers For this application QIAGEN offers the QIAGEN OneStep RT PCR Kit which enables one step RT PCR of any RNA template without optimization Two step RT PCR is generally carried out using oligo dT primers in the RT step and gene specific primers in the PCR step For the RT step QIAGEN offers two kits for efficient and sensitive reverse transcription Omniscript RT Kit for cDNA synthesis using 50 ng 2 pg RNA per reaction E Sensiscript RT Kit for cDNA synthesis using less than 50 ng RNA per reaction For the PCR step QIAGEN offers enzymes that minimize PCR optimization B Tag DNA Polymerase for PCR without a hot start E HotStarTaq DNA Polymerase for PCR with a hot start E HotStarTaq Plus DNA Polymerase for PCR with a hot start and a fast 5 minute enzyme activation time For mor
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11. 1 One bottle of Buffer RW1 350 ml contains sufficient buffer for 2 runs of 96 samples Buffer RW1 left over after a run should be stored at room temperature 15 25 for the next run Buffer RPE Before using a bottle of Buffer RPE for the first time add 4 volumes of ethanol 96 100 i e add 400 ml ethanol to 100 ml Buffer RPE Tick the check box on the label of the bottle to indicate that ethanol has been added One bottle of reconstituted Buffer RPE 500 ml contains sufficient buffer for 2 runs of 96 samples Buffer RPE left over after a run should be stored at room temperature 15 25 for the next run 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 12 RNeasy 96 BioRobot 8000 Handbook 10 2006 RNase free water For a single run of 96 samples 8 tubes of RNase free water 1 9 ml each are required Be sure to remove the lids before placing the tubes on the BioRobot worktable RNase free water left over after a run should be discarded and should not be reused for subsequent runs Top Elute Fluid For a single run of 96 samples 4 tubes of Top Elute Fluid 1 48 ml each are required Be sure to remove the lids before placing the tubes on the BioRobot worktable Top Elute Fluid left over after a run should be discarded and should not be reused for su
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13. 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 call one of the QIAGEN Technical Service Departments or local distributors see back cover RNeasy 96 BioRobot 8000 Handbook 10 2006 5 Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDSs These are available online in convenient and compact PDF format at www giagen com ts msds asp where you can find view and print the MSDS for each QIAGEN kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste Buffer RLT contains guanidine thiocyanate and Buffer RW1 contains a small amount of guanidine thiocyanate This chemical 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 If liquid containing potentially infectious agents is spilt on the BioRobot workstation clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypoc
14. NA sample before measuring purity see Appendix B page 26 RNeasy 96 BioRobot 8000 Handbook 10 2006 21 Comments and suggestions RNA degraded Inappropriate handling of starting material RNase contamination Ensure that cells have been properly handled and that the protocol has been performed without interruptions especially the initial steps involving cell lysis See Appendix A page 24 Handling and storing starting material page 12 and Important points before starting page 18 Although all buffers have been tested and are guaranteed RNase free RNases can be introduced during use Be sure not to introduce any RNases during the procedure or later handling See Appendix A page 24 Carry out periodic maintenance as described in the Maintenance environment BioRobot Universal System or BioRobot 8000 User Manual BioRobot Gene Expression Real Time RT PCR or BioRobot 8000 to prevent RNase contamination of the BioRobot workstation DNA contamination in downstream experiments 22 No DNase treatment Improper setup of DNase solutions Carry out the optional on plate DNase digestion see Appendix D page 30 Alternatively DNase digest the RNA eluates After heat inactivating the DNase the RNA samples can be used directly in downstream applications without further treatment or can be repurified see the cleanup protocols in the RNeasy MinElute Cleanup Handbook
15. October 2006 RNeasy 96 BioRobot 8000 Handbook For high throughput RNA purification from animal and human cells automated on the BioRobot Universal System BioRobot Gene Expression Real Time RT PCR or BioRobot 8000 QIAGEN Trademarks QIAGEN BioRobot HotStarTaq MinElute Omniscript QuantiTect RNeasy Sensiscript QIAGEN Group ABI PRISM Applied Biosystems Applera Corporation or its subsidiaries Agilent Agilent Technologies Inc LightCycler TaqMan Roche Group SYBR Molecular Probes Inc GlAzol Lysis Reagent is a subject of US Patent No 5 346 994 and foreign equivalents Purchase of QuantiTect SYBR Green Kits is accompanied by a limited non transferable immunity from suit to use it with detection by a dsDNA binding dye as described in U S Patents Nos 5 994 056 and 6 171 785 and corresponding patent claims outside the United States for the purchaser s own internal research No realtime apparatus or system patent rights or any other patent rights and no right to use this product for any other purpose are conveyed expressly by implication or by estoppel QuantiTect Probe Kits and QuantiTect Multiplex Kits are an Authorized 5 Nuclease Core Kit without Licensed Probe Its purchase price includes a limited non transferable immunity from suit under certain patents owned by Roche Molecular Systems Inc or F Hoffmann La Roche Ltd for using only this amount of the product in the prac
16. Troughs 20 ml cat no 9232764 Disposable Troughs 80 ml cat no 9013653 S Blocks cat 19585 96 100 ethanol and 70 ethanol in water Cell culture plates see below for recommended suppliers Optional reagents B 143 M f mercaptoethanol comercially available solutions are usually 14 3 M see protocol page 18 for details E RNase Free DNase Set cat no 79254 for optional on plate DNase digestion see Appendix D page 30 for details Screw cop tubes 2 ml for use with the optional DNase treatment Safe Lock micro test tubes Eppendorf www eppendorf com Note Use of other tubes may require modification of the QIAsoft protocol for assistance contact QIAGEN Other tubes may not fit into the reagent holder 8 tube 1 5 ml cat no 9011758 Suppliers of cell culture plates Round bottom Greiner cat no 650180 www greinerbioone com E Flatbottom Costar cat no 3599 www corning com Note Use of other cell culture plates may require modification of the QIAsoft protocol contact QIAGEN for assistance The kit contains 2 reusable S Blocks If processing several RNeasy 96 plates each day it may be convenient to have extra S Blocks Do not use denatured alcohol which contains other substances such as methanol or methylethylketone This is not complete list of suppliers and does not include many important vendors of biological supplies 10 RNeasy 96 BioRobot
17. afety data sheets 5055 available from the product supplier To make a saturated solution add solid bromophenol blue to distilled water Mix and continue to add more bromophenol blue until no more will dissolve Centrifuge to pellet the undissolved powder and carefully pipet the saturated supernatant RNeasy 96 BioRobot 8000 Handbook 10 2006 29 Appendix D Optional On Plate DNase Digestion with the RNase Free DNase Set The RNase Free DNase Set cat no 79254 provides efficient on plate digestion of DNA during RNA purification The DNase is efficiently removed in subsequent wash steps Note Standard DNase buffers are not compatible with on plate DNase digestion Use of other buffers may affect the binding of the RNA to the RNeasy silica membrane reducing the yield and integrity of the RNA Lysis and homogenization of the sample and binding of RNA to the silica 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 silica membrane The DNase is removed by a second wash with Buffer RW1 Washing with Buffer RPE and elution are then performed according to the standard protocol Important points before starting B Generally DNase digestion is not required since RNeasy 96 technology efficiently removes most of the DNA without DNase treatment However further DNA removal may be necessary for certain RNA applications that are sens
18. al plasticware if processing 192 samples in one run RNeasy 96 plate GlAplate Holder silver 6 Silver multiwell plate holder 96 well cell High speed shaker system culture plate Shaker back right Elution MP Slot 26 Blue elution microtube Microtubes CL adapter RNeasy 96 BioRobot 8000 Handbook 10 2006 15 Table 5 Loading Plasticware BioRobot Gene Expression Real Time RT PCR or BioRobot 8000 Volume for one run of ltem Position 96 samples 192 samples Ethanol Reagent holder for 3 x 20 ml troughs 70 MP Slot 13 Position 2B 17 ml 34 ml Buffer RIT Reagent holder for 3 x 20 ml troughs MP Slot 13 Position 2A 17 ml 34 ml Buffer RW1 Reagent carousel Rotor Slot 2 165 ml 271 ml Buffer RPE Reagent carousel Rotor Slot 4 251 ml 443 ml Ethanol Reagent carousel Rotor Slot 6 145 ml 190 ml 96 100 Distilled Reagent carousel Rotor Slot 8 700 ml 700 ml water RNase free Reagent holder for 8 x 2 ml tubes 8 x 1 9 ml 8 x 1 9 ml water MP Slot 12 Reagent Holder Tray Reagent holder for 8 x 2 ml tubes MP Slot 12 Reagent Holder Tray 8 x 1 9 ml Top Elute Reagent holder for 8 x 1 5 ml tubes Fluid MP Slot 12 Reagent Holder Tray A 4 1 48ml 8 1 48 ml Optional Reagent holder for 8 x 1 5 ml tubes RNase free VariTherm Slot Reagent DNase It X Holder Tray 4x 1 9 ml 8 x 1 9 ml Before using Buffer RPE for the first time be sure to add 4 volumes of ethanol 96 100 t See Appendix D page 30
19. ase digestion prepare the DNase incubation mix as described in Appendix D page 30 RNeasy 96 BioRobot 8000 Handbook 10 2006 Procedure 1 If cells have been stored at a lower temperature equilibrate them to room temperature 15 25 C Make sure that the BioRobot workstation is switched on Switch on the computer and monitor Launch the QIAsoft Operating System If using the BioRobot Universal System start the QlAsoft 5 Operating System from the Microsoft Windows Start menu where it is located under Programs QIAsoft 5 QIAsoft 5 Enter your user name and password in the Login dialog box and click OK to access QIAsoft 5 If using the BioRobot Gene Expression Real Time RT PCR or the BioRobot 8000 the QIAsoft 4 2 Operating System is required Start the software from the Microsoft Windows Start menu where it is located under Programs GlAsoft 4 2 QIAsoft 4 2 Select RNeasy 96 Total RNA from the protocol selection box This protocol is for RNA purification only If you want to perform RNA purification and RT PCR setup in the same run on the BioRobot Gene Expression Real Time RT PCR or BioRobot 8000 select RNeasy 96 RxSetup from the protocol selection box Click E to start the protocol The QlAsoft Operating System will now guide you through the remaining steps required to set up the BioRobot workstation for the RNeasy 96 BioRobot 8000 protocol Follow the steps detailed in each protocol message before pr
20. bsequent runs RNase free DNase The RNeasy 96 BioRobot 8000 procedure provides the option of performing DNase digestion during RNA purification Generally DNase digestion is not required since the procedure efficiently removes most of the DNA without the use of DNase However further DNA removal may be necessary for certain RNA applications that are sensitive to very small amounts of DNA e g real time RT PCR analysis with a low abundance target For further details see Appendix D page 30 Plasticware One RNeasy 96 plate one 96 well cell culture plate and one S Block are required for a single run of 96 samples When placing these items of plasticware on the BioRobot worktable make sure that position A1 is located at the upper left corner Discard the plasticware after use the S Block can be reused One rack of Elution Microtubes CL is required for a single run of 96 samples Be sure to keep the lid on and to place the elution microtubes rack on the blue elution microtube adapter Make sure that the bar code of the elution microtubes rack faces to the right RNeasy 96 BioRobot 8000 Handbook 10 2006 13 Summary of worktable setup Table 3 Loading Buffers and Reagents BioRobot Universal System Volume for one run of Item Position 96 samples 192 samples Ethanol Reagent holder for 5 x 80 ml 70 troughs MP Slot 9 Position A 21 ml 35 ml Buffer RIT Reagent holder for 5 x 80 ml troughs MP Slot 9 Position B 21 ml
21. commended for disruption and homogenization QlAvac 96 optional QIAGEN also offers kits for reverse transcription two step RT PCR and real time two step RT PCR For details visit www qiagen com products pcr Visit www qiagen com GeneGlobe to search for and order QuantiTect Primer Assays which are gene specific primer sets for use with this kit RNeasy 96 BioRobot 8000 Handbook 10 2006 37 Ordering Information Product Contents Cat no QuantiTect Probe RT PCR Kit for quantitative real time one step RT PCR using sequence specific probes QuantiTect Probe For 200 x 50 pl reactions 3 x 1 7 ml 204443 RT PCR Kit 200 2x Master Mix 100 pl RT Mix 2 x 2 ml RNase Free Water QuantiTect Multiplex RT PCR Kits for quantitative multiplex real time one step RT PCR using sequence specific probes QuantiTect Multiplex For 200 x 50 pl reactions 3 x 1 7 ml 204643 RT PCR Kit 200 2x Master Mix contains ROX 100 pl RT Mix 2 x 2 ml RNase Free Water QuantiTect Multiplex For 200 x 50 pl reactions 3 x 1 7 ml 204843 RT PCR NR Kit 200 2x Master Mix contains no ROX dye 100 pl RT Mix 2 x 2 ml RNase Free Water The BioRobot Universal System and BioRobot 8000 workstations are intended for research applications No claim or representation is intended for their use to provide information for the diagnosis prevention or treatment of a disease The RNeasy 96 Kit RNeasy 96 Universal Tissue Kit RNeasy 96 Un
22. e information on QIAGEN products for one step RT PCR and two step RT PCR visit www giagen com products per 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 96 BioRobot 8000 Handbook 10 2006 31 Real time RT PCR The range of QuantiTect Kits guarantee highly specific and sensitive results in real time RT PCR on any real time cycler and require no optimization of reaction and cycling conditions QuantiTect Kits are available for two step and one step RT PCR and are compatible with detection by SYBR Green dye or by sequence specific probes e g TaqMan and FRET probes Multiplex RT PCR of up to 5 targets is also possible Predesigned QuantiTect Primer Assays are primer sets for use with SYBR Green detection and are easily ordered online at www qgiagen com GeneGlobe For more information on QuantiTect Kits and Assays visit www qiagen com geneXpression Quantification on real time cyclers Quantification is based on the threshold cycle where the amplification plot crosses a defined fluorescence threshold Comparison of the threshold cycles provides a highly sensitive measure of relative template concentration in different samples Figure 1 shows an example of real time analysis using duallabeled probes in TaqMan analysis Monitoring during the early
23. eagents and the positions of the plasticware are correct o 2 p 2 7 Atthe end of the protocol follow the protocol messages which guide you through the steps to clean up the BioRobot workstation Tasks include removing reagents and plasticware cleaning the channeling block and cleaning the vacuum manifold If reusing Buffer RW and RPE in a subsequent run be sure to close the bottles Use the elution microtube caps caps for strips provided to seal the microtubes for storage Store RNA at 20 C or at 70 C If using the BioRobot Universal System a protocol is available for realtime RT PCR setup select RT PCR Reaction Setup from the protocol selection box 8 Be sure to perform daily weekly monthly and annual maintenance of the BioRobot workstation IF using the BioRobot Universal System enter the Maintenance environment to find out which maintenance procedures need to be carried out For details on how to use the Maintenance environment refer to the GlAsoft 5 Operating System User Manual If using the BioRobot Gene Expression Real Time RT PCR or BioRobot 8000 refer to the BioRobot 8000 User Manual for details about maintenance procedures For all BioRobot workstations it is particularly important to prevent RNase contamination by cleaning the tubing of the workstation with O 1 M NaOH 1 mM EDTA solution This is done during the monthly maintenance For details refer to the Maintenance environ
24. easy 96 procedure all RNA molecules longer than 200 nucleotides are purified The procedure provides an enrichment for mRNA since most RNAs lt 200 nucleotides such as 5 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 cushion where small RNAs do not sediment efficiently For purification of total RNA and microRNA from cells and tissues we recommend using the miRNeasy 96 Kit see ordering information page 37 8 RNeasy 96 BioRobot 8000 Handbook 10 2006 RNeasy 96 BioRobot 8000 Procedure Sample 3 yse Add ethanol Bind RNA Wash Elute into elution microtubes Pure total RNA RNeasy 96 BioRobot 8000 Handbook 10 2006 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 BioRobot Universal System with Application Pack Gene Expression BioRobot Gene Expression Real Time RT PCR no longer available or BioRobot 8000 see ordering information page 35 Disposable gloves Disposable Filter Tips 1100 pl cat no 9012598 Disposable
25. ely glassware can be treated with DEPC 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
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27. for RNA 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 RNeasy 96 BioRobot 8000 Handbook 10 2006 11 Handling and storing starting material RNA in cells is not protected until the sample is flash frozen or disrupted in the presence of RNase inhibiting or denaturing reagents It is therefore important that cell samples are immediately frozen and stored at 70 C or processed immediately after harvesting Otherwise unwanted changes in the gene expression profile will occur The relevant procedures should be carried out as quickly as possible After disruption in Buffer lysis buffer samples can be stored at 70 C for months S Blocks The kit contains 2 S Blocks If processing several RNeasy 96 plates per day it may be convenient to have extra S Blocks available see ordering information page 35 The S Blocks are used throughout the RNeasy 96 BioRobot 8000 procedure Be sure to empty waste from the S Blocks after use To reuse the S Blocks rinse them thoroughly with tap water incubate for 2 hours or overnight 0 1 M NaOH 1 mM EDTA rinse in distilled water and dry at 50 C Preparation of reagents and worktable Buffer RLT One bottle of Buffer RIT 220 ml contains sufficient buffer for 6 runs of 96 samples Buffer RIT left over after a run should be stored at room temperature 15 25 for the next run Buffer RW
28. form satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its performance and design If a QIAGEN product does not meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover Technical Assistance At GIAGEN 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 molecular biology and the use of GIAGEN products If you have any questions or experience any difficulties regarding the RNeasy 96 BioRobot 8000 Kit 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
29. h as phenol and or chloroform The puritied RNA is ready to use in any downstream application including E RT PCR and real time RT PCR Differential display cDNA synthesis Northern dot and slot blot analyses Primer extension Poly A RNA selection RNase S1 nuclease protection Principle and procedure The RNeasy 96 BioRobot 8000 Kit uses well established technology for high throughput RNA preparation The kit combines the selective binding properties of a silica based membrane with the speed of vacuum processing The BioRobot Universal System BioRobot Gene Expression Real Time RT PCR or BioRobot 8000 provide walkaway automation of the RNeasy 96 procedure for total RNA purification from up to 5 x 10 cells per sample The procedure starts with automated removal of the cell culture medium Cells are then lysed directly in the cell culture plate on the integrated high speed shaker of the BioRobot workstation Cell lysis is performed under highly denaturing conditions with guanidine thiocyanate to immediately inactivate RNases and ensure purification of intact RNA Ethanol is added to provide appropriate binding conditions and the samples are then applied to the wells of the RNeasy 96 plate Total RNA binds and contaminants are efficiently washed away High quality RNA is then eluted in a small volume of RNase free water ready for use in any downstream application RNeasy 96 BioRobot 8000 Handbook 10 2006 7 With the automated RN
30. hlorite followed by water The following risk and safety phrases apply to the components of the RNeasy 96 BioRobot 8000 Kit Buffer RLT Contains guanidine thiocyanate Risk and safety phrases R20 21 22 32 513 26 36 46 Buffer RW1 Contains ethanol Risk and safety phrases 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 R20 21 22 Harmful by inhalation in contact with skin and if swallowed R32 Contact with acids liberates very toxic gas 13 Keep away from food drink and animal feedingstuffs S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S36 Wear suitable protective clothing S46 If swallowed seek medical advice immediately and show this container or label 6 RNeasy 96 BioRobot 8000 Handbook 10 2006 Introduction The RNeasy 96 BioRobot 8000 Kit enables simultaneous purification of total RNA from 96 or 192 samples each containing up to 5 x 10 animal or human cells The RNeasy 96 BioRobot 8000 Kit provides efficient high throughput RNA sample preparation for research use in fields such as drug screening and basic research The RNeasy 96 BioRobot 8000 procedure replaces time consuming and tedious methods involving alcohol precipitation steps large numbers of wash steps or the use of toxic substances suc
31. il biomarker biomarkerhy shaji almaz net ae 02 2880 2916 Fax 86 21 64955468 Email order taigen com Email India Pakistan info_bj genecompany com Beijing Genetix Pakistan Microbiological Associates Thailand info_sh genecompany com Shanghai Tel 91 11 51427031 Tel 492 51 5567953 Theera Trading Co Ltd info_cd genecompany com Chengdu Fax 91 11 25419631 Fax 92 51 5514134 Tel 02 412 5672 info_gz genecompany com E mail genetix genetixbiotech com Email orderpma comsats net pk Fax 02 412 3244 Guangzhou Email theetrad samart co th Indonesia Peru Genetimes Technology Inc PT Research Biolabs Turkey Order 8008202565 62 21 5865357 INMUNOCHEM SAC Medek Medikal Ur nler S Tel 51 1 4409678 tie H Tel 86 21 54262677 E mail a ve Saglik Hizmetleri A S 3 8 3 Fax 51 1 4223701 Fax 48621 64398855 indonesia researchbiolabs com Email inmunbshemMerr comi Tel 216 302 15 80 Email order genetimes com cn JRPS 216 302 15 88 ron Email makialp med ek com Colombia Zist Baran BIORAIN Poland Tel 98 21 88066348 or Syngen Biotech Sp z o o United Arab Emirates SENTECH Genefics S Technology 98 21 88066349 071 7985850 52 A Mazowr Medical amp Chemical Tel 457 44 2519037 1 Fax 498 21 88214107 Fac 071 798 58 53 Supplies Fac 57 4 2516555 mE pp Email E mail infoGbiorain com Email infoGsyngen pl Tel 4971 4 266 1272 gerencia g
32. ior to running the gel equilibrate in 1x FA gel running buffer see composition below for at least 30 minutes 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 96 BioRobot 8000 Handbook 10 2006 RNA sample preparation for FA gel electrophoresis Add 1 volume of 5x RNA loading buffer see composition below to 4 volumes of RNA sample e g 10 pl of loading buffer and 40 pl of RNA and mix Incubate for 3 5 minutes at 65 C chill on ice and load onto the equilibrated FA gel Gel running conditions Run gel at 5 7 V cm in 1 FA gel running buffer Composition of FA gel buffers 10x FA gel buffer 200 mM 3 N morpholino propanesulfonic acid MOPS free acid 50 mM sodium acetate 10 mM EDTA pH to 7 0 with NaOH 1x FA gel running buffer 100 ml 10x FA gel buffer 20 ml 37 12 3 M formaldehyde 880 ml RNase free water 5x RNA loading buffer 16 yl saturated aqueous bromophenol blue solution 80 yl 500 mM EDTA pH 8 0 720 yl 37 12 3 M formaldehyde 2 ml 10076 glycerol 3 084 ml formamide 4 ml 10x FA gel buffer RNase free water to 10 ml Stability approximately 3 months at 4 C When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material s
33. itive to very small amounts of DNA e g real time RT PCR analysis with a low abundance target DNA can also be removed by a DNase digestion following RNA purification Donot vortex the reconstituted DNase I 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 The RNeasy 96 BioRobot 8000 procedure requires 2 RNase Free DNase Sets per 96 well plate Dissolve 2 vials of solid DNase 2 x 1500 Kunitz units in 2 x 550 pl of the RNase free water provided To avoid loss of DNase do not open the vials Inject RNase free water into the vials using an RNase free needle and syringe Mix gently by inverting the vials Do not vortex Unused DNase stock solution can be stored at 20 C for up to 9 months Thawed stock solution can be stored at 2 8 C for up to 6 weeks Do not refreeze the DNase stock solution after thawing 30 RNeasy 96 BioRobot 8000 Handbook 10 2006 Procedure D1 Add 670 pl DNase stock solution see above to 7 3 ml Buffer RDD Mix by gently inverting 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 D2 Aliquot into four 2 ml Safe Lock tubes with 1 99 ml DNase incubation
34. iversal Tissue 8000 Kit miRNeasy 96 Kit QIAGEN OneStep RT PCR Kit QuantiTect SYBR Green RT PCR Kit QuantiTect Primer Assays QuantiTect Probe RT PCR Kit and QuantiTect Multiplex RT PCR Kits are intended for research use No claim or representation is intended to provide information for the diagnosis prevention or treatment of a disease Larger kit size available please inquire t Recommended for instruments from Applied Biosystems Recommended for instruments from other suppliers 38 RNeasy 96 BioRobot 8000 Handbook 10 2006 QIAGEN Distributors and Importers Please see the back cover for contact information for your local QIAGEN office Argentina Egypt Lithuania Singapore Tecnolab S A Clinilab INTERLUX Research Biolabs Pte Lid Tel 011 4555 0010 Tel 52 57 212 Tel 370 5 2786850 Tel 6777 5366 Fax 011 4553 3331 Fax 52 57 210 Fax 370 5 2796728 Fax 6778 5177 Email info tecnolab com ar Email Clinilab link net Email spiritGinterlux lt E mail salesGresearchbiolabs com Bosnia Herzegovina Estonia Malaysia Slovak Republic MEDILINE d o o Quantum Eesti AS RESEARCH BIOLABS SDN BHD BIO CONSUIT Slovakia spol s r o Tel 386 1 830 80 40 Tel 372 7301321 Tel 603 8070 3101 Tel Fax 02 5022 1336 Fax 38618308070 Fax 372 7304310 Fax 603 8070 5101 Email bio cons cdicon sk 386 1 830 80 63 Email quantum quantum ee Email biolabs tm net my Sans
35. ked by denaturing agarose gel electrophoresis and ethidium bromide staining or using an Agilent 2100 bioanalyzer The respective ribosomal RNAs should appear as sharp bands or peaks The apparent ratio of 28S rRNA to 18S rRNA should be approximately 2 1 If the ribosomal bands or peaks of a specific sample are not sharp but appear as a smear towards smaller sized RNAs it is likely that the RNA sample suffered major degradation during preparation Appendix C Formaldehyde Agarose Gel Electrophoresis The following protocol for formaldehyde agarose FA gel electrophoresis is routinely used at QIAGEN and gives enhanced sensitivity for gel and subsequent analysis e g northern blotting A key feature is the concentrated RNA loading buffer that allows a larger volume of RNA sample to be loaded onto the gel than conventional protocols e g Sambrook J et al 1989 Molecular cloning a laboratory manual 2nd ed Cold Spring Harbor NY Cold Spring Harbor Laboratory Press FA gel preparation To prepare FA gel 1 2 agarose of size 10 x 14 x 0 7 cm mix 1 2 g agarose 10 ml 10x FA gel buffer see composition below Add RNase free water to 100 ml If smaller or larger gels are needed adjust the quantities of components proportionately Heat the mixture to melt agarose Cool to 65 C in a water bath Add 1 8 ml of 37 12 3 formaldehyde and 1 pl of a 10 mg ml ethidium bromide stock solution Mix thoroughly and pour onto gel support Pr
36. l rights reserved 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 10 Important Notes 11 Amount of cells 11 Handling and storing starting material 12 S Blocks 12 Preparation of reagents and worktable 12 Protocol El Purification of Total RNA from Animal or Human Cells 18 Troubleshooting Guide 21 Appendix A General Remarks on Handling RNA 24 Appendix B Storage Quantification and Determination of Quality of RNA 26 Appendix C Formaldehyde Agarose Gel Electrophoresis 28 Appendix D Optional On Plate DNase Digestion with the RNase Free DNase Set 30 Appendix E RT PCR and Real Time RT PCR 31 Ordering Information 35 GIAGEN Distributors and Importers 39 RNeasy 96 BioRobot 8000 Handbook 10 2006 3 Kit Contents RNeasy 96 BioRobot 8000 Kit 12 Catalog no 967152 Number of preps 12 x 96 RNeasy 96 Plates 12 Register Cards 96 well 12 S Blocks 2 Elution Microtubes CL 12 x 96 Caps for Strips 165 x 8 Buffer 2 x 220 ml Buffer RW 1 6 x 350 ml Buffer RPE concentrate 6 x 100 ml RNase Free Water 96 x 1 9 ml Top Elute Fluid 48 x 1 48 ml Handbook 1 Reusable see page 12 for cleaning instructions t Contains a guanidine salt Not compatible with disinfecting reagents containing bleach See page 6 for safe
37. lts The free sample volume in a 25 pl one tube real time RT PCR analysis is typically 9 10 pl For a 50 yl assay with approximately twice the free sample volume we recommend using 1 3 6 9 12 15 18 and 20 pl RNeasy 96 BioRobot 8000 Handbook 10 2006 33 Determination of Linear Range for TaqMan Analysis 20 19 9 x 18 5 2 E 17 16 1 2 4 6 8 10 RNeasy 96 eluate pl Figure 2 Linearity of RNeasy 96 RNA purification for one tube B actin RT PCR analysis using dual labeled TaqMan probes Total RNA was purified from 5 x 104 Hela cells with the RNeasy 96 Kit RNA was eluted in 2 x 60 pl RNase free water RT PCR TaqMan analysis of B actin mRNA was performed in triplicate using 1 2 4 6 8 and 10 pl of the RNeasy 96 eluate in a 25 pl reaction volume and the entire triplicated series was repeated three times The mean of the threshold cycle for each volume is presented here plotted against the logarithm of the volume Error bars represent the o _ standard deviation The linear response range covers all volumes from 1 to 10 pl 34 RNeasy 96 BioRobot 8000 Handbook 10 2006 Ordering Information Product Contents Cat no RNeasy 96 BioRobot For 12 x 96 total RNA preps on the 967152 8000 Kit 12 BioRobot Universal System BioRobot Gene Expression Real Time RT PCR or BioRobot 8000 12 RNeasy 96 Plates Elution Microtubes CL Caps S Blocks RNase Free Reagents and Buffers BioRobot Universal System for
38. ment or BioRobot 8000 User Manual 20 RNeasy 96 BioRobot 8000 Handbook 10 2006 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise 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 molecular biology applications see back cover for contact information Comments and suggestions Clogged plate wells Too much starting material Reduce the amount of starting material It is essential to use the correct amount of starting material see Amount of cells page 11 Little or no RNA eluted much starting material Overloading significantly reduces RNA yield Reduce the amount of starting material see Amount of cells page 11 b Buffer temperatures too low All buffers must be at room temperature 15 25 C throughout the procedure c Residual liquid in cell culture Make sure the correct layout configuration plate after removal of medium flat bottom or round bottom is entered in the Run Protocol Layout Configuration dialog box Use of plates from some suppliers may result in incomplete removal of cellculture medium See Equipment and Reagents to Be Supplied By User page 10 for recommended suppliers Low A Asso value Water used to dilute RNA for Use 10 mM Tris Cl pH 7 5 not RNase free A20 Asso measurement water to dilute the R
39. n racks of 96 includes cap strips Buffer RLT 220 ml 220 ml RNeasy Lysis Buffer 79216 Top Elute Fluid 48 x 1 48 ml 48 x 1 48 ml Top Elute Fluid 1020460 RNase Free DNase Set 50 1500 units RNase Free DNase 79254 RNase Free Buffer RDD and RNase Free Water Related products for RNA purification RNeasy 96 Universal Tissue 8000 Kit for automated high throughput RNA purification from any type of tissue RNeasy 96 Universal For 12 x 96 total RNA preps on the 967852 Tissue 8000 Kit 12 BioRobot Universal System BioRobot Gene Expression Real Time RT PCR or BioRobot 8000 12 RNeasy 96 Plates Collection Microtubes Elution Microtubes CL Caps S Blocks GlAzol Lysis Reagent RNase Free Reagents and Buffers RNeasy 96 Kit for high throughput RNA minipreps from cells RNeasy 96 Kit 4 For 4 x 96 total and cytoplasmic 74181 RNA preps 4 RNeasy 96 Plates Elution Microtubes CL Caps S Blocks AirPore Tape Sheets RNase Free Reagents and Buffers GIAGEN offers a wide range range of RNA purification kits for different sample types sizes and throughputs These include the BioRobot EZ1 and BioRobot M48 for automated RNA purification from 1 6 and 6 48 cell or tissue samples respectively For details visit www giagen com RNA Requires use of the Plate Rotor 2 x 96 and Centrifuge 4K15C Tissuelyser recommended for disruption and homogenization Larger kit size available please inquire Requires u
40. nificance Aygo readings should be greater than 0 15 An absorbance of 1 unit at 260 nm corresponds to 44 pg of RNA per milliliter A452 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 27 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 25 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 20 pl of RNA sample 180 pl of 10 mM Tris Cl pH 7 0 1 10 dilution Measure absorbance of diluted sample in a 0 2 ml cuvette RNase free Aus 0 2 Concentration of RNA sample 44 pg ml x dilution factor 44 pg ml x 0 2 x 10 88 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 26 RNeasy 96 BioRobot 8000 Handbook 10 2006
41. oceeding to the next protocol message You will be prompted to enter information for the following options Layout configuration Select the type of 96 well cell culture plate used m Number of samples Select 96 samples 1 plate protocol or 192 samples 2 plate protocol m DNase treatment Enter yes to perform DNase digestion on the RNeasy 96 plate see page 29 m Change tips Indicate if you want to change the tips during removal of culture supernatant For many applications changing tips is not necessary The cells remain intact during removal of the culture supernatant and cross contamination of RNA between samples is minimal For sensitive applications the tips can be changed so as to eliminate the possibility of cross contamination m Elution volume Choose the elution volume For most applications we recommend the default elution volume RNeasy 96 BioRobot 8000 Handbook 10 2006 19 0204014 m Automatic clog detection BioRobot Universal System only Enter yes to perform clog detection The BioRobot workstation will then check for clogged membranes on the RNeasy 96 plate Any wells with clogged membranes will not be processed further in the RNA purification procedure Note that this option requires the use of more disposable tips and increases run time If using the BioRobot Universal System a load check will be automatically performed after you have set up the workstation to check that the volumes of the r
42. on 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 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 25 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 24 RNeasy 96 BioRobot 8000 Handbook 10 2006 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 Alternativ
43. 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 t Plastics used for some electrophoresis tanks are not resistant to ethanol Take proper care and check the supplier s instructions RNeasy 96 BioRobot 8000 Handbook 10 2006 25 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 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 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 accurately quantified using an Agilent 2100 bioanalyzer quantitative RT PCR or fluorometric quantification Spectrophotometric quantification of RNA To ensure sig
44. or RNeasy 96 Handbook Make sure that the tubes of DNase incubation mix each contain 1 99 ml of DNase mix Use 2 ml Safe Lock tubes from Eppendorf Use of other tubes may require modification of the QIAsoft protocol Other tubes also may not fit in the reagent holder 8 tube 1 5 ml RNeasy 96 BioRobot 8000 Handbook 10 2006 Comments and suggestions RNA does not perform well in downstream experiments Salt carryover during elution Ensure that Buffer RPE is at room temperature 15 25 C RNeasy 96 BioRobot 8000 Handbook 10 2006 23 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 contaminati
45. r DNA removal may be desirable for certain RNA applications that are sensitive to very small amounts of DNA In these cases small residual amounts of DNA can be removed by optional on plate DNase digestion see Appendix D page 30 or by DNase digestion after RNA purification please contact QIAGEN Technical Services for a protocol Buffer RLT may form a precipitate upon storage If necessary warm to 37 C to redissolve When purifying RNA from cells containing high amounts of RNases it may be necessary to add B mercaptoethanol to Buffer RIT to avoid degradation of RNA supports the inactivation of RNases by guanidine thiocyanate Add 10 pl B ME per 1 ml Buffer RLT Dispense in a fume hood and wear appropriate protective clothing Buffer containing can be stored at room temperature 15 25 for up to 1 month In most cases it will not be necessary to add to Buffer RLT Buffer RLT and Buffer RW1 contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach See page 6 for safety information All steps of the procedure should be performed at room temperature 20 25 Avoid any interruptions 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 to obtain a working solution Check that all buffers are at room temperature 15 25 If carrying out optional on plate DN
46. se of QlAvac 96 or the QIAGEN 96 Well Plate Centrifugation system 36 RNeasy 96 BioRobot 8000 Handbook 10 2006 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 96 Kit for purification of microRNA and total RNA from a wide range of animal tissues and cells 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 GlAzol lysis Reagent RNase Free Reagents and Buffers Related products for one step RT PCR and real time one step RT PCR QIAGEN OneStep RT PCR Kit for fast and successful one step RT PCR QIAGEN OneStep For 25 x 50 pl reactions Enzyme 210210 RT PCR Kit 25 Mix 5x PCR Buffer dNTP Mix 5x Q Solution RNase Free Water QuantiTect SYBR Green RT PCR Kit for quantitative real time one step RT PCR using SYBR Green QuantiTect SYBR Green For 200 x 50 pl reactions 3 x 1 7 ml 204243 RT PCR Kit 200 2x Master Mix 100 pl RT Mix 2 x 2 ml RNase Free Water Larger kit size available please inquire Requires use of the Plate Rotor 2 x 96 and Centrifuge 4K15C Tissuelyser re
47. tice of the 5 nuclease process solely for the purchaser s own internal research when used in conjunction with Licensed Probe No right under any other patent claims such as apparatus or system claims and no right to use this product for any other purpose is hereby granted expressly by implication or by estoppel Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA Purchase of the QuantiTect SYBR Green RT PCR Kit QuantiTect Probe RT PCR Kit and QuantiTect Multiplex RT PCR Kits is accompanied by a limited non transferable license under RT and Reverse Transcription PCR patents owned by Roche Molecular Systems Inc and F Hoffmann La Roche Ltd to use it for the purchaser s own internal research No realtime patent rights of any kind no right under any other patent claims such as apparatus or system claims and no right to use this product for any other purpose is hereby granted expressly by implication or by estoppel QuantiTect Primer Assays are compatible for use in 5 nuclease process or the dsDNA binding dye processes covered by patents owned by Roche or owned by or licensed to Applera Corporation No license under these patents to practice the 5 nuclease process or the dsDNA binding dye processes are conveyed expressly by implication to the purchaser by the purchase of this product 2002 2006 QIAGEN al
48. tions so that genomic DNA will not be amplified Alternatively DNA contamination can be detected on agarose gels following RT PCR by performing control experiments in which no reverse transcriptase is added prior to the PCR step or by using intro spanning primers For sensitive applications such as differential display or if it is not practical to use splice junction primers DNase digestion of the purified RNA with RNase free DNase is recommended 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 96 BioRobot 8000 Handbook 10 2006 27 A protocol for optional on plate DNase digestion using the RNase free DNase Set is provided in Appendix D page 30 The DNase is efficiently washed away in the subsequent wash steps Alternatively after the RNeasy procedure the eluate containing the RNA can be treated with DNase please contact QIAGEN Technical Services for a protocol The RNA can then be repurified using an RNeasy RNA cleanup protocol see the RNeasy 96 Handbook or the RNeasy MinElute Cleanup Handbook or after heat inactivation of the DNase the RNA can be used directly in downstream applications Integrity of RNA The integrity and size distribution of total RNA purified with RNeasy Kits can be chec
49. ty information Add 4 volumes of ethanol 96 100 before use to obtain a working solution The following kit components are also available separately S Blocks Elution Microtubes CL including caps for strips Buffer RIT and Top Elute Fluid See page 35 for ordering information Storage The RNeasy 96 BioRobot 8000 Kit including all reagents and buffers should be stored dry at room temperature 15 25 C and is stable for at least 9 months under these conditions Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of RNeasy 96 BioRobot 8000 Kit is tested against predetermined specifications to ensure consistent product quality RNeasy 96 BioRobot 8000 Handbook 10 2006 Product Use Limitations The RNeasy 96 BioRobot 8000 Kit is intended for research use No claim or representation is intended to provide information for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to per
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