QIAamp® Viral RNA Mini Handbook
Contents
1. 3 El a e o 2 b a gt E 5 gt 28 Carefully apply 630 pl of the lysate from step 5 into the QlAamp Mini column without wetting the rim Avoid touching the GlAamp Mini column membrane with the pipet tip Open the main vacuum valve Be sure to leave the lid of QlAamp Mini column open while applying vacuum After all lysates have been drawn through the QlAamp Mini column close the main vacuum valve and open the screw cap valve to vent the manifold Close the screw cap valve after the vacuum is released from the manifold After closing the main vacuum valve the vacuum is applied only to the connecting system if used and not the vacuum manifold IF the lysates from individual samples have not completely passed through the membrane despite the VacValves of all other QlAamp Mini columns being closed place the GlAamp Mini column into a clean 2 ml collection tube not provided close the cap and centrifuge at full speed for 3 min or until it has completely passed through Continue with step 7 of the spin protocol on page 24 Additional collection tubes can be purchased separately see Ordering Information page 42 Centrifugation is performed at 6000 x g 8000 rpm in order to limit microcentrifuge noise Centrifugation at full speed will not affect the yield or purity of viral RNA Repeat steps 7 and 8 If the sample volume was greater than 140 pl repeat these steps until al2. Always use caution and wear safety glasses when working near a vacuum manifold under pressure Contact QIAGEN Technical Services or your local distributor for information concerning spare or replacement parts The vacuum pressure is the pressure differential between the inside of the vacuum manifold and the atmosphere standard atmospheric pressure 1013 millibar or 760 mm Hg and can be measured using the QlAvac Connecting System or a vacuum regulator see Figure 1 The vacuum protocol requires a vacuum pump capable of producing a vacuum of 800 to 900 mbar e g QIAGEN Vacuum Pump Higher vacuum pressures must be avoided Use of vacuum pressures lower than recommended may reduce DNA yield and purity and increase the frequency of clogged membranes QlAamp Viral RNA Mini Handbook 04 2010 19 Table 4 Chemical resistance properties of QlAvac 24 Plus Resistant to Acetic acid Chaotropic salts Chlorine bleach Chromic acid Hydrochloric acid SDS Sodium chloride Sodium hydroxide Tween 20 Urea Not resistant to Benzene Chloroform Ethers Phenol Toluene Knob Figure 1 Schematic diagram of the Vacuum Regulator 20 QlAamp Viral RNA Mini Handbook 04 2010 Regulator gauge Setup of the GlAvac 24 Plus vacuum manifold 1 Connect the QlAvac 24 Plus to a vacuum source If using the QlAvac Connecting System connect the system to the manifold and vacuum source as described in Appe
3. Close the GlAamp Mini column before placing it in the microcentrifuge Centrifuge as described E Remove the Gl amp Mini column and collection tube from the microcentrifuge Place the QlAamp Mini column a new collection tube Discard the filtrate and the old collection tube Please note that the filtrate may contain hazardous waste and should be disposed of properly E Open only one GlAamp Mini column at a time and take care to avoid generating aerosols For efficient parallel processing of multiple samples it is recommended to fill a rack with collection tubes to which the GlAamp Mini columns can be transferred after centrifugation Used collection tubes containing the filtrate can be discarded and the new collection tubes containing the GlAamp Mini columns can be placed directly in the microcentrifuge Vacuum protocol on the QlAvac 24 Plus The GlAvac 24 Plus is designed for fast and efficient vacuum processing of up to 24 GIAGEN spin columns in parallel Samples and wash solutions are drawn through the column membranes by vacuum instead of centrifugation providing greater speed and reduced hands on time in purification procedures In combination with the QlAvac Connecting System optional the QlAvac 24 Plus can be used as a flow through system The sample flow through is collected in a separate waste bottle For maintenance of the GlAvac 24 Plus please refer to the handling guidelines in the QlAvac 24 Plus Handbook
4. Using a vacuum pressure that is too high may damage the QlAamp membrane Using a vacuum pressure which is too low may cause reduced RNA yield and purity Use a vacuum regulator see ordering information on page 43 to adjust the pressure to 800 to 900 mbar for all vacuum steps QlAamp Viral RNA Mini Handbook 04 2010 35 Appendix 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 only 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 non disposable vessels and solutions while working with RNA General handling Proper microbiological aseptic technique should always be used when working with RNA Hands and dust particles may carry bacteria and molds and are the most common sources of RNase contamination Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin or from dusty laboratory equipment Change gloves frequently and keep tubes closed whenever possible During the procedure
5. 18 GlAamp Viral RNA Mini Handbook 04 2010 Processing QlAamp Mini columns on the QlAvac 24 Plus QlAamp Mini columns are processed on QlAvac 24 Plus using VacConnectors and reusable VacValves VacValves optional are inserted directly into the luer slots of the QlAvac 24 Plus manifold and ensure a steady flow rate facilitating parallel processing of samples of different natures e g plasma and serum volumes or viscosities They should be used if sample flow rates differ significantly in order to ensure consistent vacuum VacConnectors are disposable connectors that fit between GlAamp Mini columns and VacValves or between GlAamp Mini columns and the GlAvac 24 Plus manifold They prevent direct contact between the column and VacValve or GlAvac 24 Plus manifold during purification thereby avoiding any cross contamination between samples VacConnectors are discarded after a single use Handling guidelines for the QlAvac 24 Plus B Always place the GlAvac 24 Plus on a secure bench top or work area If dropped the GlAvac 24 Plus manifold may crack Always store the GlAvac 24 Plus clean and dry For cleaning procedures see the QlAvac 24 Plus Handbook The components of the QlAvac 24 Plus are not resistant to certain solvents Table 4 If these solvents are spilled on the unit rinse it thoroughly with water ensure consistent performance do not apply silicone or vacuum grease to any part of the QlAvac 24 Plus manifold
6. to Buffer AW2 concentrate as indicated on the bottle and in Table 3 Buffer AW2 is stable for 1 year when stored closed at room temperature but only until the kit expiration date Table 3 Preparation of Buffer AW2 Kit cat no No of preps AW concentrate Ethanol Final volume 52904 50 13 ml 30 ml 43 ml 52906 250 66 ml 160 ml 226 ml Contains chaotropic salt Take appropriate laboratory safety measures and wear gloves when handling Not compatible with disinfecting agents that contain bleach See page 6 for safety information t Contains sodium azide as a preservative QlAamp Viral RNA Mini Handbook 04 2010 17 Handling of QlAamp Mini columns Owing to the sensitivity of nucleic acid amplification technologies the following precautions are necessary when handling QlAamp Mini columns to avoid cross contamination between sample preparations E Carefully apply the sample or solution to the QlAamp Mini column Pipet the sample into the QlAamp Mini column without wetting the rim of the column B Change pipet tips between all liquid transfer steps The use of aerosol barrier tips is recommended Avoid touching the membrane with the pipet tip E After all pulse vortexing steps briefly centrifuge 1 5 ml microcentrifuge tubes to remove drops from the inside of the lid B Wear gloves throughout the procedure In case of contact between gloves and sample change gloves immediately Spin protocol
7. South Orders 080 000 7146 Fax 02 2626 5703 Technical 080 000 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1 122 330 Technical 01 800 7742 639 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 1800 742 4362 Fax 65 6854 8184 Technical 1800 742 4368 Spain Orders 91 630 7050 Fax 911 630 5145 Technical 9 1 630 7050 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN Sample amp Assay Technologies
8. treatment 15 min 70 C to inactivate the DNase QlAamp Viral RNA Mini Handbook 04 2010 9 Warnings and precautions RNA is extremely sensitive to RNases and should always be prepared with due care Hands and dust particles may carry bacteria and molds and are the most common sources of RNase contamination Please read Handling RNA in the Appendix page 36 of this handbook before starting PCR should always be carried out using good laboratory practices Accordingly a PCR laboratory should always be divided into three areas an area for preparation of reagents an area for preparation of samples and an area for amplification and detection Due to the high sensitivity of PCR it is absolutely necessary that all reagents remain pure and uncontaminated and should be monitored carefully and routinely Contaminated reagents must be discarded Sample volumes GlAamp Mini columns can bind RNA greater than 200 nucleotides in length Actual yield will depend on sample size sample storage and virus titer The procedure is optimized for use with 140 pl samples but samples up to 280 pl can be used Small samples should be adjusted to 140 pl with phosphate buffered saline PBS before loading and samples with a low viral titer should be concentrated to 140 pl before processing For samples larger than 140 yl the amount of lysis buffer and other reagents added to the sample before loading must be increased proportionally but the amounts of B
9. DNA Blood Mini Kit For 50 minipreps 50 GlAamp Mini 51104 50 Spin Columns QIAGEN Protease Reagents Buffers and Collection Tubes 2 ml QlAamp DNA Blood Mini Kit 250 minipreps 250 GlAamp Mini 51106 250 Spin Columns QIAGEN Protease Reagents Buffers and Collection Tubes 2 ml QlAamp 96 DNA Blood Kit for high throughput genomic DNA purification from blood and body fluids GlAamp 96 DNA Blood Kit For 4 x 96 preps 4 QlAamp 96 Plates 51161 4 QIAGEN Protease Reagents Buffers Lysis Plates and Collection Vessels QlAamp 96 DNA Blood Kit For 12 x 96 preps 12 QlAamp 96 51162 12 Plates QIAGEN Protease Reagents Buffers Lysis Plates and Collection Vessels QlAamp DNA Mini Kit for genomic DNA purification from tissue blood and body fluids GlAamp DNA Mini Kit 50 For 50 minipreps 50 GlAamp Mini 51304 Spin Columns Proteinase K Reagents Buffers and Collection Tubes 2 ml GlAamp DNA Mini Kit 250 For 250 minipreps 250 GlAamp Mini 51306 Spin Columns Proteinase K Reagents Buffers and Collection Tubes 2 ml Fully automatable on the GlAcube See www giagen com for protocols t Requires use of the QIAGEN 96 Well Plate Centrifugation System Please inquire QlAamp Viral RNA Mini Handbook 04 2010 Al Ordering Information Product Contents Cat no Accessories Buffer AW 1 concentrate 242 ml Wash Buffer 1 Concentrate 19081 for 1000 preparations Buffer AW2 concentrate 324 ml Wash Buffer 2 Concentrat
10. RNA Mini Handbook 04 2010 11 Spin and vacuum procedures The GlAamp Viral RNA Mini purification procedure is carried out in three steps using QlAamp Mini columns in a standard microcentrifuge on a vacuum manifold or on the QlAcube The procedures are designed to ensure that there is no sample to sample cross contamination and allow safe handling of potentially infectious samples QlAamp Mini columns fit into most standard microcentrifuge tubes In the spin protocol due to the volume of filtrate 2 ml collection tubes provided are required to support QlAamp Mini column during loading and wash steps For the vacuum protocol a vacuum manifold GlAvac 24 Plus or equivalent see page 19 and a vacuum pump capable of producing a vacuum of 800 to 900 mbar e g QIAGEN Vacuum Pump are required Eluted RNA can be collected in standard 1 5 ml microcentrifuge tubes not provided These tubes must be RNase free to avoid degradation of viral RNA by RNases Automated viral RNA purification on the QlAcube Purification of viral RNA using the QlAamp Viral RNA Mini Kit can be fully automated on the QlAcube The innovative QlAcube uses advanced technology fo process QIAGEN spin columns enabling seamless integration of automated low throughput sample prep into your laboratory workflow The QlAcube performs the same steps as the manual procedure lyse bind wash and elute enabling you to continue using the GlAamp Viral RNA Mini Kit for purificat
11. RNase free Buffers QIAGEN Protease Elution Microtubes Caps S Blocks Carrier RNA EZ1 Virus Mini Kit for automated simultaneous purification of viral DNA and RNA from 1 6 serum and plasma samples using EZ1 workstations EZ1 Virus Mini Kit 48 For 48 virus nucleic acid preps 955338 Reagent Cartridges Disposable Tips Disposable Tip Holders Sample Tubes Elution Tubes Buffers EZ1 Virus Card Preprogrammed card for EZ1 virus 9016386 protocols MagAttraci Virus Mini M48 Kit for automated simultaneous purification of viral DNA and RNA from serum and plasma using the BioRobot M48 workstation MagAttract Virus Mini For 192 virus nucleic acid preps 955336 M48 Kit 192 MagAttract Suspension B and RNase Free Reagents and Buffers App Package M48 Software protocol package for 9016145 Infectious Disease infectious disease applications v 2 0 on the BioRobot M48 workstation GlAamp RNA Blood Mini Kit for total RNA purification from blood and body fluids QlAamp RNA Blood For 50 RNA preps 50 GlAamp Mini 52304 Mini Kit 50 Spin Columns 50 GlAshredder Spin Columns Collection Tubes 1 5 ml and 2 ml RNase free Reagents and Buffers Wash buffers are labeled with bar codes and expiration date is stated on the Q card in the kit 40 GlAamp Viral RNA Mini Handbook 04 2010 Ordering Information Product Contents Cat no GlAamp DNA Blood Mini Kit for genomic DNA purification from blood and body fluids GlAamp
12. and the relevant protocol sheet Larger starting volumes up to 560 yl in multiples of 140 pl can be processed by increasing the initial volumes proportionally and loading the GlAamp Mini column multiple times as described below in the protocol Some samples with very low viral titers should be concentrated before the purification procedure see Protocol Sample Concentration page 30 Alternatively larger sample volumes can be processed using one of the following kits which provide simultaneous purification of viral DNA and RNA B QlAamp MinElute Spin Kit 200 pl B GlAamp MinElute Vacuum Kit 500 pl B QlAamp UltraSens Virus Kit 1000 pl Important points before starting E Read Important Notes pages 15 22 before starting the protocol All centrifugation steps are carried out at room temperature 15 25 Things to do before starting E Equilibrate samples to room temperature 15 25 E Eqmuilibrate Buffer AVE to room temperature for elution in step 11 M Check that Buffer AW1 and Buffer AW2 have been prepared according to the instructions on page 17 E Add carrier RNA reconstituted in Buffer AVE to Buffer AVL according to instructions on page 15 Procedure 1 Pipet 560 pl of prepared Buffer AVL containing carrier RNA into 1 5 ml microcentrifuge tube IF the sample volume is larger than 140 yl increase the amount of Buffer AVL carrier RNA proportionally e g 280 pl sample will require 1120 pl Buf
13. described on page 15 This solution should be prepared fresh and is stable at 2 8 C for up to 48 hours Buffer AVL carrier RNA develops a precipitate when stored at 2 8 C that must be redissolved by warming at 80 C before use Unused portions of carrier RNA dissolved in Buffer AVE should be frozen in aliquots at 20 C Do not freeze thaw the aliquots of carrier RNA more than 3 times DO NOT warm Buffer AVL carrier RNA solution more than 6 times DO NOT incubate at 80 for more than 5 min Frequent warming and extended incubation will cause degradation of the carrier RNA leading to reduced recovery of viral RNA and eventually false negative RT PCR results particularly when low titer samples are used 4 GlAamp Viral RNA Mini Handbook 04 2010 Product Use Limitations The GlAamp Viral RNA Mini Kit is intended for molecular biology applications This product is not intended for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We recommend all users of GIAGEN 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 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 sat
14. instructions to a final volume of 140 yl Some samples plasma in particular may be difficult to concentrate to 140 yl due to high viscosity Centrifugation for up to 6 hours may be necessary 3 Pipet 140 pl of concentrated sample into a 1 5 ml microcentrifuge tube and follow the Viral RNA Mini Spin Protocol on page 23 QlAamp Viral RNA Mini Handbook 04 2010 31 gt gt 8 3 5 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 Little or no RNA in the eluate d 9 32 Carrier RNA not added to Buffer AVL Degraded carrier RNA Sample frozen and thawed more than once Low concentration of virus in the sample Inefficient protein denaturation in Buffer Buffer AVL prepared incorrectly No ethanol added to the lysate step 5 Low percentage ethanol used Comments and suggestions Reconstitute carrier RNA in Buffer AVE and mix with Buffer AVL as described on page 15 Repeot the purification procedure
15. load the lysate onto the QGlAamp Mini column in several steps Removal of residual contaminants Viral RNA bound to the QlAamp membrane is washed free of contaminants during two short centrifugation or vacuum steps The use of two different wash buffers AW 1 and AW2 significantly improves the purity of the eluted RNA Optimized wash conditions ensure complete removal of any residual contaminants without affecting RNA binding Elution with Buffer AVE Buffer AVE is RNase free water that contains 0 0476 sodium azide to prevent microbial growth and subsequent contamination with RNases Sodium azide affects spectrophotometric absorbance readings between 220 and 280 nm but has no effect on downstream applications such as RT PCR Should you wish to determine the purity of the eluted RNA elution with RNase free water instead of Buffer AVE is recommended Cellular DNA contamination The GlAamp Viral RNA Mini Kit is not designed to separate viral RNA from cellular DNA and both will be purified in parallel if present in the sample To avoid copurification of cellular DNA the use of cell free body fluids for preparation of viral RNA is recommended Samples containing cells such as cerebrospinal fluid bone marrow urine and most swabs should first be filtered or centrifuged for 10 minutes at 1500 x g and the supernatant used If RNA and DNA have been isolated in parallel the eluate can be DNase digested using RNase free DNase followed by heat
16. minutes to remove residual DEPC Note Corex tubes should be rendered RNase free by treatment with DEPC and not by baking This will reduce the failure rate of this type of tube during centrifugation Electrophoresis tanks Electrophoresis tanks should be cleaned with detergent solution e g 0 5 SDS rinsed with water dried with ethanol and then filled with a solution of 3 H O After 10 minutes at room temperature the electrophoresis tanks should be rinsed thoroughly with RNase free water Solutions Solutions water and other solutions should be treated with 0 1 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 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 Trace amounts of DEPC will modify purine residues in RNA by carbethoxylation Carbethoxylated RNA is translated with very low efficiency in cell free systems However its ability to form DNA RNA or RNA RNA hybrids is not seriously affected unless a large fraction of the purine residues have been modified Residual DEPC must
17. with the kit The concentration of carrier RNA has been adjusted so that the QlAamp Viral RNA Mini Kit can be used as a generic purification system compatible with many different amplification systems and is suitable for a wide range of RNA viruses Different amplification systems vary in efficiency depending on the total amount of nucleic acid present in the reaction Eluates from this kit contain both viral nucleic acids and carrier RNA and amounts of carrier RNA will greatly exceed amounts of viral nucleic acids Calculations of how much eluate to add to downstream amplifications should therefore be based on the amount of carrier RNA added To obtain the highest levels of sensitivity in amplification reactions it may be necessary to adjust the amount of carrier RNA added to Buffer AVL Addition of internal controls Using the QlAamp Viral RNA Mini protocols in combination with commercially available amplification systems may require the introduction of an internal control into the purification procedure Internal control RNA or DNA should be added together with the carrier RNA to the lysis buffer For optimal purification efficiency internal control molecules should be longer than 200 nucleotides as smaller molecules are not efficiently recovered Refer to the manufacturer s instructions in order to determine the optimal concentration Using a concentration other than that recommended may reduce amplification efficiency QlAamp Viral
18. 30 For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www qiagen com or can be requested from QIAGEN Technical Services or your local distributor Japan US and Canada Rest of world QlAamp Viral RNA Mini Handbook 04 2010 43 www qiagen com Australia Orders 1 800 243 800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 86 21 3865 3865 Fax 86 21 3865 3965 Technical 800 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 800 789 544 Fax 02 334304 826 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea
19. AVL Briefly centrifuge the tube to remove drops from the inside of the lid Add 560 pl of ethanol 96 100 to the sample and mix by pulse vortexing for 15 s After mixing briefly centrifuge the tube to remove drops from inside the lid Insert a QlAamp Mini column into the VacConnector on the QlAvac 24 Plus vacuum manifold Only ethanol should be used since other alcohols may result in reduced yield and purity of the RNA Do not use denatured alcohol which contains other substances such as methanol or methylethylketone If the sample volume is greater than 140 increase the amount of ethanol proportionally e g a 280 pl sample will require 1120 pl of ethanol In order to ensure efficient binding it is essential that the sample is mixed thoroughly with the ethanol to yield a homogeneous solution The collection tube from the blister pack can be saved for the centrifugation in step 14 Make sure that the main vacuum valve between the vacuum pump and the vacuum manifold and the screw cap valve on the end of the QlAvac 24 Plus vacuum manifold are closed Switch on the vacuum pump by pressing the power switch The vacuum is applied only to the connecting system if used and not to the vacuum manifold Note For fast and convenient release of the vacuum pressure the QlAvac Connecting System or the Vacuum Regulator should be used see Ordering Information page 43 QlAamp Viral RNA Mini Handbook 04 2010 27 lt Q e
20. RNA due to temperature change before start of process Repeat the procedure with new samples and ensure that no precipitate has formed in Buffer AVL carrier RNA at the beginning of the process Ensure that carrier RNA has been reconstituted in Buffer AVE and added to Buffer AVL see page 15 Determine the maximum amount of carrier RNA suitable for your RT PCR Adjust the concentration of carrier RNA added to Buffer AVL accordingly QlAamp Viral RNA Mini Handbook 04 2010 33 9 Reduced sensitivity Buffers AW1 and AW2 used in the wrong order New combination of reverse transcriptase and Taq DNA polymerase used DNA contamination DNA and RNA present in the sample General handling 34 Lysate not completely passed through the membrane Comments and suggestions Determine the maximum volume of eluate suitable for your RT PCR Reduce the volume of eluate added to the RT PCR Ensure that Buffer AW1 and Buffer AW2 are used in the correct order in the protocol Repeat the purification procedure with a new sample If enzymes are changed it may be necessary to readjust the amount of carrier RNA solution added to Buffer AVL To avoid copurification of DNA use of cell free body fluids for preparation of viral RNA is recommended Samples containing cells such as cerebrospinal fluid bone marrow urine and most swabs should be made cell free by centrifugation or filtration If using centrifugation p
21. Third Edition April 2010 GlAamp Viral RNA Mini Handbook For purification of viral RNA from plasma serum cell free body fluids cell culture supernatants Trademarks QIAGEN GlAamp GlAcube BioRobof EZ1 MagAttract MinElute UltraSens QIAGEN Group Centricon UltraFree Millipore Corex Corning Inc Tween ICI Americas Inc 1999 2007 QIAGEN all rights reserved JS FSC Wwenilscorg QIAGEN is a member of the Forest Stewardship Council FSC For production of printed materials Papler aus ver antwortungsvollen including handbooks GIAGEN has a policy to select suppliers that comply with FSC standards for printing processes and well managed forests Contents Kit Contents 4 Storage 4 Product Use Limitations 5 Product Warranty and Satisfaction Guarantee 5 Technical Assistance 5 Quality Control 6 Safety Information 6 Introduction 7 Principle 7 Procedure 7 Cellular DNA contamination 9 Warnings and precautions 10 Sample volumes 10 Lysis 10 Carrier RNA 1 Addition of internal controls 11 Spin and vacuum procedures 12 Automated viral RNA purification on QlAcube 12 Determination of yield 13 Determination of viral RNA length 13 Equipment and Reagents to Be Supplied by User 14 Important Notes 15 Preparation of reagents 15 Handling of GlAamp Mini columns 18 Handling guidelines for the QlAvac 24 Plus 19 Centrifugation 22 Proto
22. always be removed from solutions or vessels by autoclaving or heating to 100 C for 15 minutes Add 0 1 ml DEPC to 100 ml of the solution to be treated Shake vigorously to bring the DEPC into solution or let the solution bake for 12 hours at 37 C Autoclave for 15 minutes to remove any trace of DEPC It may be desirable to test water sources for the presence of contaminating RNases since many sources of distilled water are free of RNase activity Note GlAamp Viral RNA buffers are not rendered RNase free by 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 5055 available from the product supplier Plastics used for some electrophoresis tanks are not resistant to ethanol Take proper care and check the supplier s instructions QlAamp Viral RNA Mini Handbook 04 2010 37 Ordering Information Product Contents Cat no GlAamp Viral RNA Mini Kit for viral RNA purification from plasma serum and cell free body fluids GlAamp Viral RNA Mini Kit For 50 minipreps 50 GlAamp Mini 52904 50 Spin Columns Carrier RNA Buffers and Collection Tubes 2 ml GlAamp Viral RNA Mini Kit For 250 minipreps 250 GlAamp Mini 52906 250 Spin Columns Carrier RNA Buffers and Collection Tubes 2 ml GlAcube and GlAcube accessories for fully autom
23. ated sample preparation using QIAGEN spin column kits QlAcube 110 V Robotic workstation for automated 9001292 QlAcube 230 V purification of DNA RNA or proteins 9001293 using QIAGEN spin column kits 1 year warranty on parts and labor Warranty PLUS 2 Full 3 year warranty 48 hour 2 working 9240834 QlAcube days priority response all labor travel and repair parts Starter Pack QlAcube Pack includes reagent bottle racks 3 990395 rack labeling strips 8 200 pl filter tips 1024 1000 pl filter tips 1024 1000 pl filter tips wide bore 1024 30 ml reagent bottles 18 rotor adapters 240 rotor adapter holder Filter Tips 1000 pl 1024 Sterile Disposable Filter Tips racked 990352 Rotor Adapters 10 x 24 For 240 preps 240 Disposable Rotor 990394 Adapters for use with the GlAcube QlAamp Viral RNA Mini Additional Buffers and Reagents 1048147 Accessory Set for use with at least 11 x QlAamp Viral RNA Mini Kits 50 catalog number 52904 or 5 x QlAamp Viral RNA Mini Kits 250 catalog number 52906 on the QlAcube US Canada and Japan Rest of world 38 GlAamp Viral RNA Mini Handbook 04 2010 Ordering Information Product Contents Cat no Related products GlAamp Virus Kits for simultaneous purification of viral RNA and DNA from plasma and serum GlAamp MinElute Virus For 50 minipreps 50 GlAamp MinElute 57704 Spin Kit 50 Columns QIAGEN Protease Carrier RNA Buffers Collect
24. cols Purification of Viral RNA Spin Protocol 23 E Purification of Viral RNA Vacuum Protocol 26 8 Sample Concentration 30 Purification of Cellular Bacterial or Viral DNA from Urine 31 Troubleshooting Guide 32 Appendix 36 Ordering Information 38 QlAamp Viral RNA Mini Handbook 04 2010 3 Kit Contents GlAamp Viral RNA Kit 50 250 Catalog no 52904 52906 Number of preps 50 250 QlAamp Mini Spin Columns 50 250 Collection Tubes 2 ml 200 1000 Buffer AVL 31 ml 5x31 ml Buffer AW1 concentrate 19 ml 95 ml Buffer AW2 concentrate 13 ml 66 ml Buffer AVE 3 x2 ml 10 x 2 ml Carrier RNA poly A 310 pg 5x 310 yg Handbook 1 1 Contains chaotropic salt which is an irritant Not compatible with disinfecting reagents which contain bleach See page 6 for safety information Contains sodium azide as a preservative Storage GlAamp Mini spin columns should be stored dry at room temperature 15 25 storage at higher temperatures should be avoided All solutions should be stored at room temperature unless otherwise stated GlAamp Mini spin columns and all buffers and reagents can be stored under these conditions until the expiration date on the kit box without showing any reduction in performance Lyophilized carrier RNA can be stored at room temperature 15 25 until the expiration date on the kit box Carrier RNA should be dissolved in Buffer AVE dissolved carrier RNA should be immediately added to Buffer AVL as
25. e 19072 for 1000 preparations Buffer AVL 5 x 31 ml Viral Lysis Buffer and 19073 5 x 310 pg Carrier RNA for 250 preparations Buffer AL 216 ml for 1000 preparations 19075 Buffer ATL 200 ml Tissue Lysis Buffer for 1000 19076 preparations Buffer AE 240 ml Elution Buffer for 1000 19077 preparations Buffer AVE 108 x 2 ml 1020953 Carrier RNA 12 x 1350 pg 1017647 Collection Tubes 2 ml 1000 Collection Tubes 2 ml 19201 GIAGEN Protease 125 mg 40 45 mAU mg lyophilized 19155 GIAGEN Protease 4 x 125 mg 40 45 mAU mg IS lyophilized Protease Resuspension Buffer 1020952 Protease Solvent 1021055 QIAGEN Proteinase K 2 2 ml gt 600 mAU nml solution 19131 QIAGEN Proteinase K 10 10 ml 600 mAU ml solution 19133 QlAvac 24 Plus Vacuum manifold for processing 19413 1 24 spin columns includes QlAvac 24 Plus Vacuum Manifold Luer Plugs Quick Couplings VacConnectors 500 500 disposable connectors for use 19407 with GlAamp spin columns on luer connectors VacValves 24 24 valves for use with the GlAvac 24 19408 and GlAvac 24 Plus 42 GlAamp Viral RNA Mini Handbook 04 2010 Ordering Information Product Contents Cat no Vacuum Pump Universal vacuum pump capacity 84000 34 L min 8 mbar vacuum abs 84010 84020 GlAvac Connecting System System to connect vacuum manifold 19419 with vacuum pump includes Tray Waste Bottles Tubing Couplings Valve Gauge 24 VacValves Vacuum Regulator For use with GlAvac manifolds 195
26. e researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please see our Technical Support center at www qiagen com goto TechSupportCenter or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www qiagen com QlAamp Viral RNA Mini Handbook 04 2010 5 Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of GlAamp Viral RNA Mini Kits is tested against predetermined specifications to ensure consistent product quality Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDSs These are available online in convenient and compact PDF format at www qiagen com support MSDS aspx where you can find view and print the MSDS for each QIAGEN kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to waste containing Buffer AVL or Buffer AW1 Buffers AVL and AW1 contain guanidine salts which can form highly reactive com pounds 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 affec
27. ellet the cells for 10 min at 1500 x g and use supernatant for isolation of viral RNA If DNA free RNA is required digest either the sample or the eluate with RNase free DNase DNase in the eluate must be inactivated by heat treatment 15 min 70 C Using spin protocol Centrifuge for 1 min at full speed or until all the lysate has passed through the membrane Using vacuum protocol Insufficient vacuum was applied or the lid of the spin column was closed during the vacuum step Increase the vacuum and open the lid while applying the vacuum If the vacuum pressure cannot be increased place the QlAamp Mini column in a clean 2 ml collection tube close the cap and centrifuge at 6000 x g 8000 rpm for 3 min or until the lysate has completely passed through the membrane Place the QlAamp Mini column into another clean 2 ml collection tube and discard the tube containing the filtrate Continue with step 7 of the spin protocol on page 24 GlAamp Viral RNA Mini Handbook 04 2010 b Clogged membrane c Cross contamination between samples d Vacuum pressure too high too low Comments and suggestions Cryoprecipitates have formed in plasma due to repeated freezing and thawing Do not use plasma that has been frozen and thawed more than once To avoid cross contamination when handling GlAamp Mini spin columns follow the guidelines in Handling of GlAamp Mini columns on page 18 Repeat the purification procedure with new samples
28. ethanol In order to ensure efficient binding it is essential that the sample is mixed thoroughly with the ethanol to yield a homogeneous solution Carefully apply 630 pl of the solution from step 5 to the QlAamp Mini column in a 2 ml collection tube without wetting the rim Close the cap and centrifuge at 6000 g 8000 rpm for 1 min Place the QlAamp Mini column into a clean 2 ml collection tube and discard the tube containing the filtrate Close each spin column in order to avoid cross contamination during centrifugation Centrifugation is performed at 6000 x g 8000 rpm in order to limit microcentrifuge noise Centrifugation at full speed will not affect the yield or purity of the viral RNA If the solution has not completely passed through the membrane centrifuge again at a higher speed until all of the solution has passed through Carefully open the GlAamp Mini column and repeat step 6 IF the sample volume was greater than 140 pl repeat this step until all of the lysate has been loaded onto the spin column Carefully open GlAamp Mini column and add 500 pl of Buffer AW1 Close the cap and centrifuge at 6000 x g 8000 rpm for 1 min Place the QlAamp Mini column in a clean 2 ml collection tube provided and discard the tube containing the filtrate It is not necessary to increase the volume of Buffer AW even if the original sample volume was larger than 140 yl GlAamp Viral RNA Mini Handbook 04 2010 9 Carefu
29. fer AVL carrier RNA and use a larger tube Fully automatable on GlAcube See www qiagen com MyQlAcube for protocols QlAamp Viral RNA Mini Handbook 04 2010 23 J020104q o 2 b a o 24 Add 140 pl plasma serum urine cell culture supernatant or cell free body fluid to the Buffer AVL carrier RNA in the microcentrifuge tube Mix by pulse vortexing for 15 s To ensure efficient lysis it is essential that the sample is mixed thoroughly with Buffer AVL to yield a homogeneous solution Frozen samples that have only been thawed once can also be used Incubate at room temperature 15 25 for 10 min Viral particle lysis is complete after lysis for 10 min at room temperature Longer incubation times have no effect on the yield or quality of the purified RNA Potentially infectious agents and RNases are inactivated in Buffer AVL Briefly centrifuge the tube to remove drops from the inside of the lid Add 560 yl of ethanol 96 100 to the sample and mix by pulse vortexing for 15 s After mixing briefly centrifuge the tube to remove drops from inside the lid Only ethanol should be used since other alcohols may result in reduced RNA yield and purity Do not use denatured alcohol which contains other substances such as methanol or methylethylketone If the sample volume is greater than 140 increase the amount of ethanol proportionally e g a 280 pl sample will require 1120 pl of
30. filtrate Carefully open the GlAamp Mini spin column Add 60 pl of Buffer AVE equilibrated to room temperature Close the cap and incubate at room temperature for 1 min Centrifuge at 6000 x g 8000 rpm for 1 min A single elution with 60 pl of Buffer AVE is sufficient to elute at least 9076 of the viral RNA from the QlAamp Mini spin column Performing a double elution using 2 x 40 yl of Buffer AVE will increase yield by up to 10 Elution with volumes of less than 30 yl will lead to reduced yields and will not increase the final concen tration of RNA in the eluate Viral RNA is stable for up to one year when stored at 20 C or 70 lt Q e 3 O o e e QlAamp Viral RNA Mini Handbook 04 2010 29 Protocol Sample Concentration Plasma serum urine cerebrospinal fluid bone marrow and other body fluids often have very low viral titers In these cases concentrating samples of up to 3 5 ml to a final volume of 140 pl is recommended Important point before starting Use centrifugal microconcentrators such as Centricon 100 Amicon 2 ml cat no 4211 Microsep 100 Filtron 3 5 ml cat no OD100CA0O Ultrafree CL Millipore 2 ml cat no UFC4 THK 25 or equivalent from other suppliers Procedure 1 Apply up to 3 5 ml of sample to the microconcentrator following the manufacturer s instructions 2 Centrifuge according to manvfacturer s instructions to a final volume of 140 yl Some sample
31. for reliable use in amplification technologies Viral RNA can be purified from plasma treated with anticoagulants other than heparin serum and other cell free body fluids Samples may be fresh or frozen but if frozen should not be thawed more than once Repeated freeze thawing of plasma samples will lead to reduced viral titers and should be avoided for optimal sensitivity Cryoprecipitates accumulate when samples are subjected to repeated freeze thawing cycles This may lead to clogging of the QlAamp membrane when using the vacuum protocol GlAamp Viral RNA Mini Kits are general purpose kits which can be used for isolation of viral RNA from a wide variety of viruses but performance can not be guaranteed for every virus Procedure QlAamp Viral RNA Mini Kits represent a well established general purpose technology for viral RNA preparation The kit combines the selective binding properties of a silica gel based membrane with the speed of microspin or vacuum technology and is ideally suited for simultaneous processing of multiple samples QlAamp Viral RNA spin protocols can be fully automated on the QlAcube The sample is first lysed under highly denaturing conditions to inactivate RNases and to ensure isolation of intact viral RNA Buffering conditions are then adjusted to provide optimum binding of the RNA to the QlAamp membrane and the sample is loaded onto the QlAamp Mini spin column The RNA binds to the membrane and contaminants are eff
32. has been shown to be better for your amplification system transfer only the required amount of dissolved carrier RNA to the tubes containing Buffer AVL Use of less than 5 6 pg carrier RNA per sample must be validated for each par ticular sample type and downstream assay Buffer AVL carrier RNA should be prepared fresh and is stable at 2 8 C for up to 48 hours This solution develops a precipitate when stored at 2 8 C that must be redis solved by warming at 80 C before use Do not warm Buffer AVL carrier RNA solution more than times Do not incubate at 80 for more than 5 minutes Frequent warm ing and extended incubation will cause degradation of carrier RNA leading to reduced recovery of viral RNA and eventually false negative RT PCR results This is particularly the case with low titer samples 16 GlAamp Viral RNA Mini Handbook 04 2010 Buffer AW1 Buffer AW1 is supplied as a concentrate Before using for the first time add the appropriate amount of ethanol 96 100 as indicated on the bottle and in Table 2 Buffer AW is stable for 1 year when stored closed at room temperature but only until the kit expiration date Table 2 Preparation of Buffer AW1 Kit cat no No of preps AW1 concentrate Ethanol Final volume 52904 50 19 ml 25 ml 44 ml 52906 250 95 ml 125 ml 220 ml Buffer AW2 Buffer AW2 is supplied as a concentrate Before using for the first time add the appropriate amount of ethanol 96 100
33. ibrate Buffer AVE to room temperature for elution in step 10 Check that Buffer AW1 and Buffer AW2 have been prepared according to the instructions on page 17 Add carrier RNA reconstituted in Buffer AVE to Buffer AVL according to instructions on page 15 For processing using VacConnectors and VacValves set up QlAvac 24 Plus as described on page 21 Fully automatable on the QlAcube See www giagen com MyQlAcube for protocols 26 GlAamp Viral RNA Mini Handbook 04 2010 Procedure 1 Pipet 560 pl of prepared Buffer AVL containing carrier RNA into 1 5 ml microcentrifuge tube IF the sample volume is larger than 140 yl increase the amount of Buffer AVL carrier RNA proportionally e g a 280 pl sample will require 1120 pl Buffer AVL carrier RNA and use a larger tube Add 140 pl plasma serum urine cell culture supernatant or cell free body fluid to the Buffer AVL carrier RNA in the microcentrifuge tube Mix by pulse vortexing for 15 s To ensure efficient lysis it is essential that the sample is mixed thoroughly with Buffer AVL to yield a homogeneous solution Frozen samples that have only been thawed once can also be used Incubate at room temperature 15 25 for 10 min Viral particle lysis is complete after lysis for 10 min at room temperature Longer incubation times have no effect on the yield or quality of the purified RNA Potentially infectious agents and RNases are inactivated in Buffer
34. iciently washed away in two steps using two different wash buffers High quality RNA is eluted in a special RNase free buffer ready for direct use or safe storage The purified RNA is free of protein nucleases and other contaminants and inhibitors The special GlAamp membrane guarantees extremely high recovery of pure intact RNA in just twenty minutes without the use of phenol chloroform extraction or alcohol precipitation All buffers and reagents are guaranteed to be RNase free QlAamp Viral RNA Mini Handbook 04 2010 7 eqnoyqo uo 4 GlAamp Viral RNA QlAamp Viral RNA Mini Spin Procedure Mini Vacuum Procedure Sample Sample Lyse 10 Bind amp Vacuum Wash Buffer AW1 CES Vacuum Wash Buffer AW2 Vacuum Elute Pure viral nucleic acid GlAamp Viral RNA Mini Handbook 04 2010 Adsorption to the QlAamp membrane The buffering conditions of the lysate must be adjusted to provide optimum binding conditions for the viral RNA before loading the sample onto the GlAamp Mini column Viral RNA is adsorbed onto the QlAamp silica membrane during two brief centrifugation steps or by vacuum Salt and pH conditions in the lysate ensure that protein and other contaminants which can inhibit downstream enzymatic reactions are not retained on the GlAamp membrane If the initial sample volume is larger than 140 yl it will be necessary to
35. ing 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 Ethanol 96 1005 1 5 ml microcentrifuge tubes Sterile RNase free pipet tips pipet tips with aerosol barriers for preventing cross contamination are recommended Microcentrifuge with rotor for 1 5 ml and 2 ml tubes For vacuum protocols QlAvac 24 Plus vacuum manifold cat no 19413 or equivalent VacConnectors cat no 19407 Vacuum Regulator cat 19530 for easy monitoring of vacuum pressures and easy releasing of vacuum Vacuum Pump cat no 84010 USA and Canada 84000 Japan or 84020 rest of world or equivalent pump capable of producing a vacuum of 800 to 900 mbar Optional VacValves cat no 19408 Optional GlAvac Connecting System cat no 19419 Do not use denatured alcohol which contains other substances such as methanol or methylethylketone 14 GlAamp Viral RNA Mini Handbook 04 2010 Important Notes IF preparing RNA for the first time please read Handling RNA in the Appendix of this handbook page 36 All steps of the GlAamp Viral RNA Mini protocols should be performed quickly and at room temperature After collection and centrifugation plasma untreated or treated with anticoagulants other than heparin or serum can be stored at 2 8 C for up to 6 hours For long term st
36. ion Tubes 2 ml QlAamp MinElute Virus For 50 minipreps 50 QlAamp 57714 Vacuum Kit 50 MinElute Columns QIAGEN Protease Carrier RNA Buffers Extension Tubes 3 ml Collection Tubes 1 5 ml QlAamp UltraSens Virus Kit For 50 viral nucleic acid preps 53704 50 50 GlAamp Mini Spin Columns Proteinase K Carrier RNA Collection Tubes 2 ml Buffers QlAamp UltraSens Virus Kit For 250 viral nucleic acid preps 53706 250 250 QlAamp Mini Spin Columns Proteinase K Carrier RNA Collection Tubes 2 ml Buffers QlAamp MinElute Media Kit for purification of DNA from liquid media QlAamp MinElute Media Kit For 50 minipreps 50 GlAamp MinElute 57414 Columns QIAGEN Proteinase K Carrier RNA Buffers Extension Tubes 3 ml Collection Tubes 1 5 ml QlAamp Media MDx Kit for automated purification of DNA from liquid media using the BioRobot MDx workstation GlAamp Media MDx Kit For 12 x 96 preps 12 GlAamp 965752 12 96 Plates Buffers Proteinase K S Blocks Disposable Troughs Racks with Elution Microtubes CL 0 4 ml Carrier RNA Top Elute Fluid Caps Tape Pad Fully automatable on GlAcube See www qiagen com MyQlAcube for protocols GlAamp Viral RNA Mini Handbook 04 2010 39 Ordering Information Product Contents Cat no GlAamp Virus BioRobot Kits for automated high throughput purification of viral RNA and DNA from plasma and serum GlAamp Virus BioRobot For 12 x 96 preps 12 GlAamp 965652 MDx Kit 12 96 Plates
37. ion of high quality viral RNA The QlAcube is preinstalled with protocols for purification of plasmid DNA genomic DNA RNA viral nucleic acids and proteins plus DNA and RNA cleanup The range of protocols available is continually expanding and additional QIAGEN protocols can be downloaded free of charge at www giagen com MyQlAcube Detailed protocols for using the QlAamp Viral RNA Mini Kit on the QlAcube are provided with the QlAcube 12 GlAamp Viral RNA Mini Handbook 04 2010 Automated viral RNA purification Viral RNA purification using the GlAamp Viral RNA Mini Kit can be fully automated on the QlAcube Determination of yield Yields of viral RNA isolated from biological samples are normally less than 1 pg and therefore difficult to determine photometrically Keep in mind that the carrier RNA 5 6 140 pl sample will account for most of the RNA present Quantitative RT PCR is recommended for determination of viral RNA yield Determination of viral RNA length The size distribution of viral RNA purified using QlAamp spin columns can be checked by denaturing agarose gel electrophoresis followed by hybridization with a virus specific labeled probe and autoradiography Sambrook J and Russell D W 2001 Molecular Cloning A Laboratory Manual 3rd ed Cold Spring Harbor NY Cold Spring Harbor Laboratory Press QlAamp Viral RNA Mini Handbook 04 2010 13 Equipment and Reagents to Be Supplied by User When work
38. isfactorily due to any reason other than misuse GIAGEN 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 We will credit your account or exchange the product as you wish Separate conditions apply to GIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover or visit www 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 the GlAamp Viral RNA Mini Kit or GIAGEN 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 th
39. ith disinfecting agents containing bleach See page 6 for safety information QlAamp Viral RNA Mini Handbook 04 2010 21 Figure 2 Setting up the QlAvac 24 Plus with GlAamp Mini columns using VacValves and VacConnectors GlAvac 24 Plus vacuum manifold slot of the 24 Plus VacValve optional VacConnector GlAamp column Luer slot closed with luer plug Must be purchased separately Centrifugation GlAamp Mini columns will fit into most standard 1 5 ml or 2 ml microcentrifuge tubes Additional 2 ml collection tubes are available separately Centrifugation of GlAamp Mini columns is performed at 6000 x g 8000 rpm in order to limit centrifuge noise Centrifugation at full speed will not affect RNA yield Centrifugation at lower speeds for lysate loading and the first wash step is also acceptable provided that the complete solution is transferred through the membrane At the second wash step centrifugation at full speed is strongly recommended All centrifugation steps are carried out at room temperature 22 GlAamp Viral RNA Mini Handbook 04 2010 Protocol Purification of Viral RNA Spin Protocol This protocol is for purification of viral RNA from 140 pl plasma serum urine cell culture media or cellfree body fluids using a microcentrifuge For automated purification of viral RNA using the GlAamp Viral RNA Mini Kit on QlAcube refer to the GlAcube User Manual
40. l of the lysate has been drawn through the GlAamp Mini column Apply 750 pl of Buffer AW1 to the QlAamp Mini column without wetting the rim Avoid touching the GlAamp Mini column membrane with the pipet tip It is not necessary to increase the volume of Buffer AW even if the original sample volume was larger than 140 yl Open the main vacuum valve After all Buffer AW1 has been drawn through the QlAamp Mini column close the main vacuum valve and open the screw cap valve to vent the manifold Close the screw cap valve after the vacuum is released from the manifold Apply 750 pl of Buffer AW2 to the QlAamp Mini column without wetting the rim Avoid touching the QlAamp Mini column membrane with the pipet tip Leave the lid of the column open Open the main vacuum valve After all Buffer AW2 has been drawn through the QlAamp Mini column close the main vacuum valve and open the screw cap valve to vent the manifold Close the screw cap valve after the vacuum is released from the manifold QlAamp Viral RNA Mini Handbook 04 2010 14 Close the lid of the QlAamp Mini column Remove it from the vacuum manifold and discard the VacConnector Place the QlAamp Mini spin column in a clean 2 ml collection tube saved from step 5 and centrifuge at full speed for 1 min to dry the membrane completely 15 Place the GlAamp Mini spin column into a clean 1 5 ml microcentrifuge tube not provided Discard the collection tube containing the
41. late the volume of Buffer AVL carrier RNA mix needed per batch of samples by selecting the number of samples to be simultaneously processed from Table 1 For larger numbers of samples volumes can be calculated using the following sample calculation n x 0 56 ml y ml y ml x 10 pl ml z pl Contains chaotropic salt Take appropriate laboratory safety measures and wear gloves when handling Not compatible with disinfecting agents that contain bleach See page 6 for safety information QlAamp Viral RNA Mini Handbook 04 2010 15 where n number of samples to be processed simultaneously y calculated volume of Buffer AVL z volume of carrier RNA Buffer AVE to add to Buffer AVL Gently mix by inverting the tube 10 times To avoid foaming do not vortex Table 1 Volumes of Buffer AVL and carrier RNA Buffer AVE mix required for the QlAamp Viral RNA Mini procedure No Vol Buffer Vol carrier No Vol Buffer Vol carrier samples AVL ml RNA AVE pl samples AVL ml RNA AVE pl 0 56 5 6 13 7 28 72 8 2 1 12 V1 2 14 7 84 78 4 3 1 68 16 8 15 8 40 84 0 4 2 24 22 4 16 8 96 89 6 5 2 80 28 0 17 9 52 95 2 6 3 36 33 6 18 10 08 100 8 7 3 92 39 2 19 10 64 106 4 8 4 48 44 8 20 11 20 0102 09 9 5 04 50 4 21 11 76 117 6 10 5 60 56 0 22 12 92 123 2 11 6 16 61 6 23 12 88 128 8 12 6 72 67 2 24 13 44 134 4 Note The sample preparation procedure is optimized for 5 6 pg of carrier RNA per sample If less carrier RNA
42. lly open the GlAamp Mini column and add 500 pl of Buffer AW2 Close the cap and centrifuge at full speed 20 000 x g 14 000 rpm for 3 min Continue directly with step 11 or to eliminate any chance of possible Buffer AW2 carryover perform step 10 and then continue with step 11 Note Residual Buffer AW2 in the eluate may cause problems in downstream applications Some centrifuge rotors may vibrate upon deceleration resulting in flow through containing Buffer AW2 contacting the GlAamp Mini column Removing the GlAamp Mini column and collection tube from the rotor may also cause flow through to come into contact with the GlAamp Mini column In these cases the optional step 10 should be performed J020104q 10 Recommended Place QlAamp Mini column a new 2 ml collection tube not provided and discard the old collection tube with the filtrate Centrifuge at full speed for 1 min 11 Place the GlAamp Mini column in a clean 1 5 ml microcentrifuge tube not provided Discard the old collection tube containing the filtrate Carefully open the QlAamp Mini column and add 60 yl of Buffer AVE equilibrated to room temperature Close the cap and incubate at room temperature for 1 min Centrifuge at 6000 x g 8000 rpm for 1 min A single elution with 60 pl of Buffer AVE is sufficient to elute at least 90 of the viral RNA from the GlAamp Mini column Performing a double elution using 2 x 40 yl of Buffer AVE will increase yield b
43. ndix A of the QlAvac 24 Plus Handbook Recommended Insert a VacValve into each luer slot of the QlAvac 24 Plus that is to be used see Figure 2 VacValves should be used if flow rates of samples differ significantly to ensure consistent vacuum Insert a VacConnector into each VacValve see Figure 2 or directly into each luer slot of the QlAvac 24 Plus that is to be used Close unused luer slots with luer plugs or close the inserted VacValve Perform this step directly before starting the purification to avoid exposure of VacConnectors to potential contaminants in the air Place the GlAamp Mini columns into the VacConnectors on the manifold see Figure 2 For nucleic acid purification follow the instructions in the vacuum protocol Discard the VacConnectors appropriately after use Leave the lid of the QlAamp Mini column open while applying vacuum Switch off the vacuum between steps to ensure that a consistent even vacuum is applied during processing For faster vacuum release a vacuum regulator should be used see Figure 1 Note Each VacValve can be closed individually when the sample is completely drawn through the spin column allowing parallel processing of samples of different volumes or viscosities After processing samples clean the QlAvac 24 Plus see Cleaning and Decontaminating the QlAvac 24 Plus in the QlAvac 24 Plus Handbook Note Buffers AVL and AW used in QlAamp Viral RNA Mini procedure are not compatible w
44. orage freezing at 20 C to 80 C in aliquots is recommended Frozen plasma or serum samples must not be thawed more than once Repeated freezing and thawing leads to denaturation and precipitation of proteins causing reduced viral titers and subsequently reduced yields of the isolated viral RNA In addition cryoprecipitates formed by freeze thawing will cause clogging of the QlAamp membrane If cryoprecipitates are visible they can be pelleted by briefly centrifuging at 6800 x g for 3 minutes The cleared supernatant should be removed without disturbing the pellet and processed immediately This step will not reduce viral titers The GlAamp Viral RNA Mini procedure is not designed to separate RNA from DNA To avoid cellular DNA contamination follow the guidelines in Cellular DNA contamination on page 10 of this handbook The QlAamp Viral RNA Mini procedure isolates all RNA molecules larger than 200 nucleotides Smaller RNA molecules will not bind quantitatively under the conditions used Preparation of reagents Addition of carrier RNA to Buffer AVL Add 310 pl Buffer AVE to the tube containing 310 pg lyophilized carrier RNA to obtain a solution of 1 pg pl Dissolve the carrier RNA thoroughly divide it into conveniently sized aliquots and store it at 20 C Do not freeze thaw the aliquots of carrier RNA more than 3 times Check Buffer AVL for precipitate and if necessary incubate at 80 C until the precipi tate is dissolved Calcu
45. s plasma in particular may be difficult to concentrate to 140 yl due to high viscosity Centrifugation for up to 6 hours may be necessary 3 Pipet 140 pl of concentrated sample into a 1 5 ml microcentrifuge tube and follow the QlAamp Viral RNA Mini Spin Protocol on page 23 2 iz o v 2 o 30 GlAamp Viral RNA Mini Handbook 04 2010 Protocol Purification of Cellular Bacterial or Viral DNA from Urine Buffer AVL used in the GlAamp Viral RNA Mini procedure inactivates the numerous unidentified PCR inhibitors found in urine For isolation of cellular bacterial or viral DNA from urine for use in PCR the QlAamp Viral RNA Mini Spin Protocol page 23 is recommended Urine often contains very low numbers of cells bacteria or viruses In these cases we recommend concentrating samples of up to 3 5 ml to a final volume of 140 yl as described below before processing For purification of DNA from Gram positive bacteria please contact QIAGEN Technical Services Important point before starting Use centrifugal microconcentrators such as Centricon 100 Amicon 2 ml cat no 4211 Microsep 100 Filtron 3 5 ml cat no OD100C40 Ultrafree CL Millipore 2 ml cat no UFC4 THK 25 or equivalent from other suppliers Procedure 1 Apply up to 3 5 ml of sample to the microconcentrator following the manvfacturer s instructions 2 Centrifuge according to manvfacturer s
46. suggestions We strictly recommend the use of ethanol as isopropanol causes reduced yields Often RNA is degraded by RNases in the starting material plasma serum body fluids Ensure that the samples are processed quickly If necessary add RNase inhibitor to the sample Check for RNase contamination of buffers and water and ensure that no RNase is introduced during the procedure Discard contaminated Buffer AVE Repeat the purification procedure with a new sample and a fresh tube of Buffer AVE Additional Buffer AVE is available separately see page 42 for ordering information Check that Buffer AW1 and AW2 concentrates were diluted with correct volumes of pure 96 1007 ethanol Do not use denatured alcohol which contains other substances such as methanol or methylethylketone Repeat the purification procedure with a new sample Check that Buffer AW1 and AW2 concentrates were diluted with 96 100 ethanol Repeat the purification procedure with a new sample Ensure that Buffer AW and Buffer AW2 are used the correct order in the protocol Repeat the purification procedure with a new sample RNA does not perform well in subsequent enzymatic reactions Little or no RNA in the eluate b Inefficient virus lysis in Buffer AVL c Buffer AVL prepared incorrectly d Too much carrier RNA in the eluate Check Little or no RNA in the eluate above for possible reasons Precipitate formed in Buffer AVL carrier
47. ted area first with laboratory detergent and water and then with 1 v v sodium hypochlorite The following risk and safety phrases apply to components of the GlAamp Viral RNA Mini Kit Buffer AVL Contains guanidine thiocyanate harmful Risk and safety phrases R20 21 22 32 13 26 36 46 Buffer AW1 Contains guanidine hydrochloride harmful irritant Risk and safety phrases R22 36 38 513 26 36 46 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 R20 21 22 Harmful by inhalation in contact with skin and if swallowed R22 Harmful if swallowed R32 Contact with acids liberates very toxic gas R36 38 Irritating to eyes and skin S13 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 36 Wear suitable protective clothing S46 If swallowed seek medical advice immediately and show container or label 6 GlAamp Viral RNA Mini Handbook 04 2010 Introduction Please take a few moments to read this handbook carefully before beginning your preparation The Important Notes on page 15 and the comments within the GlAamp Viral RNA Mini protocols beginning on page 23 are particularly valuable Principle GlAamp Viral RNA Mini Kits provide the fastest and easiest way to purify viral RNA
48. uffers AW1 and AW2 used in the wash steps usually do not need to be increased If the initial sample volume is increased application of the lysed sample to the GlAamp Mini column will require multiple loading steps There is no danger of overloading the GlAamp Mini column and the quality of the purified RNA will be unaffected For volumes greater than 560 yl concentration of the sample is recommended See Protocol Sample Concentration page 30 Lysis The sample is first lysed under the highly denaturing conditions provided by Buffer AVL to inactivate RNases and to ensure isolation of intact viral RNA Carrier RNA added to Buffer AVL improves the binding of viral RNA to the QlAamp membrane especially in the case of low titer samples and limits possible degradation of the viral RNA due to any residual RNase activity 10 GlAamp Viral RNA Mini Handbook 04 2010 Carrier RNA Carrier RNA serves two purposes Firstly it enhances binding of viral nucleic acids to the GlAamp Mini membrane especially if there are very few target molecules in the sample Secondly the addition of large amounts of carrier RNA reduces the chance of viral RNA degradation in the rare event that RNase molecules escape denaturation by the chaotropic salts and detergent in Buffer AVL If carrier RNA is not added to Buffer AVL this may lead to reduced viral RNA recovery The amount of lyophilized carrier RNA provided is sufficient for the volume of Buffer AVL supplied
49. with new samples Carrier RNA reconstituted in Buffer AVE was not stored at 20 C or underwent multiple freeze thaw cycles Alternatively Buffer AVL carrier RNA mixture was stored for more than 48 hours at 2 8 C Prepare a new tube of carrier RNA dissolved in Buffer AVE and mix with Buffer AVL Repeat the purification procedure with new samples Repeated freezing and thawing should be avoided Always use fresh samples or samples thawed only once Concentrate the sample volume to 140 pl using a microconcentrator Repeat the RNA purification procedure with a new sample See Protocol Sample Concentration on page 30 Precipitate formed in Buffer AVL carrier RNA after storage at 2 8 C was not redissolved by heating before starting the procedure Redissolve the precipitate and repeat the procedure with a new sample Check Buffer AVL for precipitate Dissolve precipitate by incubation at 80 Repeat the purification procedure with a new sample Repeat the purification procedure with a new sample Use 96 100 ethanol in step 5 Do not use denatured alcohol which contains other substances such as methanol or methylethylketone GlAamp Viral RNA Mini Handbook 04 2010 i Isopropanol used instead of ethanol ij degraded RNase contamination in Buffer AVE Buffer AW1 or AW2 prepared incorrectly m Buffer AW1 or AW2 prepared with 70 ethanol n Buffers AW1 and AW2 used in the wrong order Comments and
50. work quickly to avoid degradation of RNA by endogenous or residual RNases 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 Non disposable plasticware Non disposable 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 37 Alternatively chloroform resistant plasticware can be rinsed with chloroform to inactivate RNases Glassware Glassware should be treated before use to ensure that it is RNase free Glassware used for RNA work should be cleaned with detergent thoroughly rinsed and oven baked at gt 240 C for four or more hours overnight if more convenient before use Autoclaving alone will not fully inactivate many RNases Oven baking will inactivate 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 36 GlAamp Viral RNA Mini Handbook 04 2010 ribonucleases Alternatively glassware can be treated with DEPC diethyl pyrocarbonate Rinse the glassware with 0 1 DEPC 0 1 in water overnight 12 hours at 37 C and then autoclave or heat to 100 C for 15
51. y up to 1076 Elution with volumes of less than 30 yl will lead to reduced yields and will not increase the final concentration of RNA in the eluate Viral RNA is stable for up to one year when stored at 20 C or 70 C QlAamp Viral RNA Mini Handbook 04 2010 25 2 e b a E 2 5 gt Protocol Purification of Viral RNA Vacuum Protocol This protocol is for purification of viral RNA from 140 pl plasma serum urine cell culture media or cell free body fluids using the GlAvac 24 Plus or equivalent vacuum manifold Larger starting volumes up to 560 yl in multiples of 140 pl can be processed by increasing the initial volumes proportionally and loading QlAamp Mini spin column multiple times as described below in the protocol Some samples with very low viral titers should be concentrated before the purification procedure see Protocol Sample Concentration page 30 Alternatively larger sample volumes can be processed using one of the following kits which provide simultaneous purification of viral DNA and RNA B QlAamp MinElute Spin Kit 200 yl QlAamp MinElute Vacuum Kit 500 pl B QlAamp UltraSens Virus Kit 1000 yl Important points before starting E Read Important Notes pages 15 22 before starting the protocol All centrifugation steps are carried out at room temperature 15 25 Things to do before starting Equilibrate samples to room temperature 15 25 Equil
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