Sample & Assay Technologies QIAxcel RNA Handbook
Contents
1. zo 1922 purified PCR A 05 hda 20x For Help press F1 H 4 04 PM QIAxcel RNA Handbook 01 2008 19 Manual alignment procedure 1 Open the Parameter setup dialog box by clicking on Analysis and then Parameters in the BioCalculator menu bar 2 Inthe Data Smoothing filter pts drop down menu select 25 Check First peak in the Markers section 4 Check Apply to all documents and then click OK to apply the settings to all windows Clicking OK without checking the Apply to all documents box will apply the settings only to the active window 5 Select Analysis and then Run in the BioCalculator menu bar y If the peaks bands are not aligned refer to the troubleshooting guide in the QlAxcel User Manual for further help Parameters setup dialog box Parameter setup Retrieve 2 2 aj imum Distance 0 025000 Suspend Integration i 2 00 QuickSave 80 000000 Store di AJ 2l 3 vi Open Save Default Markers Iv First peak Last peak v Apply to all documents Use as default Determination of 28s 18s rRNA ratio 1 Open the Parameter setup dialog box by clicking on Analysis and then Parameters in the BioCalculator menu bar 2 Change the Suspend Integration value to 4 3 In the Markers panel uncheck the First peak and Last peak boxes 20 QlAxcel RNA Handbook 012. 2008 4 In the Data Smoothing filter pts drop down menu select 50 and then click OK 5 Select Analysis and then Run in the BioCalculator menu bar The results table will display the RNA ratio Note The ratio of rRNA peaks 28S 18S in total RNA analysis can vary from 0 7 to 2 5 depending on the individual RNA sample In addition to the ratio it is recommended that visual analysis should be done to check for RNA degradation Degraded RNA samples should have multiple peaks between 18S and 288 Additional peaks should also be evident before the 18S band If extraneous peaks are detected they may be manually excluded from the analysis by right clicking on the peak and selecting Delete peak see QIAxcel User Manual Appendix D Parameters setup dialog box Parameter setup a QuickSave 80 0000 Store x 21 E E 10 0000 Retrieve ao 23 vi vw New Open see 5 Data smoothing filter pts 50 v en Default Markers Marker Display Deta H NORMAL Last peak Iv Apply to all documents Use as default Apply Cancel RNA Concentration 1 Open the Reference Markers dialog box by clicking on Analysis and then Reference Markers in the BioCalculator menu bar 2 Inthe drop down menu change the setting from OFF to Conc 3 Two new fields will appear in the dialog box Reference Markers dialog box QIAxcel RNA Hand
3. Fax 8002 2073 Technical 8002 2067 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 South Korea Orders 1544 7145 Fax 1544 7146 Technical 1544 7145 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 d at Sample amp Assay Technologies
4. calibration should be performed see the QlAxcel User Manual section 5 4 for more information This step is not necessary if the QlAxcel Gel Cartridge has already been calibrated unless it is being used on a different QlAxcel instrument or a different computer is used to operate the instrument Important The 12 tube strip should fit loosely in the MARKER2 position on the buffer tray Sample preparation recommendations The minimum sample volume required for analysis is 10 ul Less than 0 1 ul of the sample will be loaded onto the QlAxcel Gel Cartridge for analysis Eoo i SSSaSa S _S _ _ _ _ _ _ _ _ _ SSS gt _ _ _ s QIAxcel RNA Handbook 01 2008 13 Typical sample preparation procedure 1 Dilute RNA in nuclease free water as detailed in Table 1 Table 1 Suggested RNA concentrations RNA sample type Suggested concentration Total RNA 0 5 1 ug ul cRNA or single stranded RNA 100 500 ng ul Fragmented cRNA or cDNA 250 500 ng ul 2 Pipet 1 pl total RNA sample lt 1 ug pl into a 0 2 ml 12 Tube Strip 3 Add an equal volume of RNA loading buffer e g Sigma cat no 1386 4 Heat the solution at 70 C for 2 min on a heating block or PCR machine 5 Centrifuge briefly to collect any condensation 6 Bring the total sample volume to 10 pl using QX RNA Dilution Buffer and mix the solution by gently pipetting up and down a few times 7 Analyze the samples immediately Note If less than 12 sample
5. performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its performance and design If a QIAGEN product does not meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover or visit www aiagen com QlAxcel RNA Handbook 01 2008 5 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 QlAxcel RNA Quality
6. Control Kit or QIAGEN products in general please do not hesitate to contact us QIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please see our Technical Support Center at www giagen com goto TechSupportCenter or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www qiagen com QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in M Purification of DNA RNA and proteins M Nucleic acid and protein assays E microRNA research and RNAi E Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com 6 QlAxcel RNA Handbook 01 2008 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
7. E Optional Create a reference marker table before running samples see see Appendix A page 19 for more details This can be done after the sample run if preferred Procedure 1 Switch on the QlAxcel system at the power switch 2 Switch on the computer and launch the BioCalculator software 3 Install the QlAxcel Gel Cartridge See the QlAxcel User Manual for more details 4 Load the buffer tray containing the QX Alignment Marker into the buffer tray holder See the QlAxcel User Manual for more details Note If being used for the first time the QlAxcel Gel Cartridge will require calibration see page 13 Note QX Alignment Markers should be replaced every 15 20 runs or 3 days whichever comes first QX Alignment Marker should be stored at 20 C between uses QIAxcel RNA Handbook 01 2008 15 5 Load the sample strips in position A or a 96 well plate onto the sample tray holder Note The cartridge door and sample door of the QlAxcel system must remain closed during operation of the instrument Opening the cartridge door or sample door during operation will cause the system to stop any action it is currently performing 6 Select the appropriate method in the Instrument Control window Refer to Appendix C of the QlAxcel User Manual for more details on the preinstalled methods Instrument Control panel MA Instrument Control File Sequence Method Sequence Name pe Notes Meth
8. January 2008 QIAxcel RNA Handbook QlAxcel RNA Quality Control Kit For automated quantitative and qualitative RNA analysis using the QlAxcel system QIAGEN Sample amp Assay Technologies Trademarks QIAGEN QIAGEN Group The QIAxcel RNA Quality Control Kit is intended for research only Not for use in diagnostic procedures 2008 QIAGEN all rights reserved Contents Kit Contents 4 Storage 5 Quality Control 5 Product Use Limitations 5 Product Warranty and Satisfaction Guarantee 5 Technical Assistance 6 QIAGEN Sample and Assay Technologies 6 Safety Information 7 Introduction 8 Principle and procedure 8 Equipment and Reagents to Be Supplied by User 10 Important Notes 11 Preparing the QlAxcel Gel Cartridge and buffer tray 11 Sample preparation recommendations 13 Method Selection 14 Protocol E Determining RNA Quality and Quantity Using the QIAxcel RNA Quality Control Kit with the QlAxcel System 15 Troubleshooting 18 Appendix A Data Analysis 19 Aligning the gel image 19 Determination of 28s 18s rRNA ratio 20 RNA Concentration 21 Appendix B General Remarks on Handling RNA 23 Ordering Information 25 QlAxcel RNA Handbook 01 2008 3 Kit Contents QlAxcel RNA Quality Control Kit 1200 Catalog no 929102 Number of assays 12 x 100 QlAxcel RNA Quality Control Cartridge with smart key QX Separation Buffer 100 ml QX Wash Buffer 40 ml QX Mineral Cil 50 ml QX RNA Dilution Buffer 15 ml QX Intens
9. at no 929703 or 96 well plates M RNA loading buffer e g Sigma cat no 1386 E RNase free water M Disposable gloves Note If working with RNA for the first time read Appendix B page 23 Ma n e 10 QlAxcel RNA Handbook 01 2008 Important Notes Preparing the QlAxcel Gel Cartridge and buffer tray Important points before starting M The volume of buffer supplied is sufficient for 100 runs of 12 samples If required additional buffers can be purchased separately see Ordering Information page 25 M The 0 2 ml 12 tube strips containing QX Alignment Marker and QX Intensity Calibration Marker if required should fit loosely in the MARKERT and MARKER2 position see step 13 Tightly fitting tube strips may cause injection problems and damage the cartridge capillary E QX Alignment Markers should be replaced every 15 20 runs or 3 days whichever comes first Additional markers and buffers may need to be purchased see Ordering Information page 25 M When not in use the 12 tube strip containing QX Alignment Marker should be stored at 20 C Things to do before starting E If preprepared the 12 tube strip containing QX Alignment Marker should be equilibrated to room temperature 15 25 C and centrifuged briefly before use M If the QlAxcel Gel Cartridge is being used for the first time intensity calibration should be performed see step 12 and the QlAxcel User Manual section 5 4 This step is not necessary
10. book 01 2008 21 Reference Markers Filename S Total normalized area fo 3533 Conc Total concentration ng ul Il 000 22 QlAxcel RNA Handbook 01 2008 4 Inthe Total normalized area field enter the total normalized area of the known RNA samples such as RNA ladder marker or commercial total RNA Note The total normalized area na can be entered either in standard form e g 0 3655 or scientific format e g 3 655E 001 The na value for each channel is displayed in the results table 5 Inthe Total concentration ng pl field enter the total concentration of the known RNA sample 6 Click OK to apply the new settings 7 Open an individual data file and select Analysis and then Run in the BioCalculator menu bar The RNA concentration will appear in the results table Appendix B 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 dispo
11. e X CU a 8 QlAxcel RNA Handbook 01 2008 The QlAxcel system offers a number of advantages over traditional slab gel electrophoresis including S Higher sensitivity Less sample wastage minimal sample input volumes Fast analysis of up to 96 samples Automation Positive charge uei Gel matrix with dye Capillary Nucleic acid with dye LED light source O Figure 1 Sample separation process using the QlAxcel system Nucleic acid molecules are size separated by applying an electrical current to a gel filled capillary A detector in the QlAxcel instrument detects the nucleic acid molecules as they migrate towards the positively charged terminus of the capillary This data are passed through a photomultiplier before being converted to an electropherogram and gel image by the BioCalculator software Note data shown are for DNA similar data are obtained for RNA QlAxcel RNA Handbook 01 2008 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 For all protocols Wi Pipets with sterile RNase free tips M Centrifuge with rotor suitable for 0 2 ml tubes and or 96 well plates M 12 tube strips e g QX 0 2 ml 12 Tube Strip c
12. ed Customized methods can also be created contact QIAGEN Technical Services for more details The BioCalculator software supplied with the QlAxcel instrument provides both electropherogram and gel view profiles of the nucleic acid separation QlAxcel DNA High Resolution Kit QlAxcel DNA Screening Kit and QlAxcel Large Fragment Kit are also available for use with the QlAxcel system see Ordering Information page 25 These kits allow fast qualitative and quantitative analysis of DNA fragments Principle and procedure The QlAxcel system uses capillary gel electrophoresis to enable fast size based separation of nucleic acids Unlike traditional agarose gel electrophoresis the separation is performed in a capillary of a precast gel cartridge The samples are automatically loaded into an individual capillary and a voltage is applied The negatively charged nucleic acid molecules migrate through the capillary to the positively charged terminus Figure 1 As with agarose gel electrophoresis low molecular weight molecules migrate faster than high molecular weight molecules As the molecules migrate though the capillary they pass a detector which detects and measures the fluorescent signal A photomultiplier converts the fluorescent signal into electronic data which are then transferred to the computer workstation for further processing using the BioCalculator software After processing the data are displayed as an electropherogram or gel imag
13. if the QlAxcel Gel Cartridge has already been calibrated unless it is being used on a different QlAxcel instrument or a different computer is used to operate the instrument QIAxcel RNA Handbook 01 2008 11 Procedure Unpacking the QlAxcel Gel Cartridge 1 Remove all buffer bottles from the kit box 2 Remove the QlAxcel Gel Cartridge from its packaging and carefully wipe off any soft gel debris from the capillary tips using a soft tissue 3 Remove the purge cap seal from the back of the QlAxcel Gel Cartridge and place it in the QX Cartridge Stand Note A soft tissue should be used to wipe off any gel that may have leaked from the port 4 Park the cartridge at the cartridge stand reservoirs containing 10ml wash buffer covered with 2ml mineral oil Note Ensure that the capillary tips are submerged in QX Wash Buffer 5 New cartridges should be allowed to stabilize in the QX Cartridge Stand for 20 minutes prior to use Preparing the buffer tray 6 Allow all reagents to equilibrate to room temperature before use 7 Wash the buffer tray with mild detergent and hot water and rinse thoroughly with deionized water or water subjected to reverse osmosis 8 Fill the WP and WI positions of the buffer tray with 8 ml QX Wash Buffer 9 Fill the BUF position of the buffer tray with 18 ml QX Separation Buffer 10 Add mineral oil to cover all 3 positions to prevent evaporation Add 2 ml mineral oil to positions WP and WI and add 4 ml
14. ity Calibration Marker 600 ul QX 0 2 ml 12 Tube Strips 2 QX Colored 0 2 ml 12 Tube Strips 2 QX RNA Alignment Marker 1 5 ml Handbook Contains sodium azide as a preservative 4 QlAxcel RNA Handbook 01 2008 Storage All components QlAxcel RNA Quality Control Kit including gel cartridges can be stored dry at room temperature 15 25 C for up to 6 months If being used on a daily basis store the QlAxcel Gel Cartridge in the QlAxcel instrument in the Park position see page 11 If only used periodically store the QlAxcel Gel Cartridge in the cartridge stand with the capillary tips submerged in QX Wash Buffer see page 11 For long term storage cover the cartridge purge hole with the original purge cap seal and place the cartridge in its original packaging Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of QlAxcel RNA Quality Control Kits is tested against predetermined specifications to ensure consistent product quality Product Use Limitations The QIAxcel RNA Quality Control Kit is intended for research only Not for use in diagnostic procedures 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
15. mineral oil to position BUF 11 Insert the buffer tray into the buffer tray holder with the slots for the 12 tube strips facing the front of the instrument Preparing QX Alignment Marker 12 Load 15 pl QX RNA Alignment Marker into each well of a QX 0 2 ml 12 Tube Strip 13 Add 1 drop of mineral oil to each well and insert the strip into the MARKER position of the buffer tray Important The 12 tube strip should fit loosely in the MARKERT position on the buffer tray 12 QlAxcel RNA Handbook 01 2008 Installing a QlAxcel Gel Cartridge and smart key 14 Remove the QIAxcel Gel Cartridge from the QX Cartridge Stand 15 Open the cartridge door and insert the QlAxcel Gel Cartridge into the QIAxcel system The cartridge description label should be facing towards the front and the purge hole should be towards the rear of the system 16 Insert the smart key into the smart key socket The smart key can be inserted in either direction 17 Close the cartridge door The cartridge ID and cartridge type will be displayed automatically in the Instrument Control window Note The system will not recognize the cartridge and will not operate if the smart key is not inserted Intensity calibration 18 Load 15 pl QX Intensity Calibration Marker into each well of a colored QX Colored 0 2 ml 12 Tube Strip and insert it into the MARKER2 position of the buffer tray Note If the QlAxcel Gel Cartridge is being used for the first time intensity
16. nding dialog box fields 11 Recommended Click the Sample Info button to enter sample information for each well Alternatively sample information previously set up in a spreadsheet can be imported in csv file format 12 Make sure that the separation channels to be used are checked i e if running only a few samples just check those channels which are to be used Note Unused wells should contain QX RNA Dilution Buffer to prevent damage to the channel 13 Check Create gel image window at start of acquisition 14 Check Automatically analyze after data acquisition 15 Check Include reference marker table optional 16 Click on the Marker button and open the desired RNA concentration table 17 Check the status of the QlAxcel system in the Status Panel Make sure the cartridge door CD and sample door SD are closed Note The Status Panel is at the bottom of the Instrument Control window and displays information on the status of the QlAxcel system see QlAxcel User Manual for more details 18 Click Run to start sample processing At the start of the run a window containing an electropherogram and a gel image will open see Appendix A page 19 QIAxcel RNA Handbook 01 2008 17 Troubleshooting The QlAxcel User Manual contains a troubleshooting guide which may be helpful in solving any problems that may arise see QlAxcel User Manual Section 8 In addition extensi
17. od 1E Sample Pes Time Runs Inc LILILILILILILIL X XI lt I lt I Xl lt I I 4 120 80 5 YON FANN 3273 30 Adjust separation time 0 L pa D sec Sample Info iv Method directory C Program Files BioCalculator Methods Vv Local data directory C Program FilessBioCalculatorD ata S Network data directory E User ID V Create gel image window at start of acquisition pom lv Automatically analyze after data acquisition Plate ID Autoscale time axis during acquisition Cartridge ID Include reference marker table Markers Cart Latch Cart Unlatch Park Change Buffer idle COM SD Closed CD Closed Prest OK Pres2 OK Latched RunsleftQ 16 QlAxcel RNA Handbook 01 2008 7 Enter the sample name position and number of runs in the relevant fields of the Instrument Control window 8 In the time column enter the sample injection time minimum 5 s maximum 40 s When left blank the default settings for the method chosen are used 9 To perform multiple analyses of the same row enter the number of repeats in the Runs field To run a 96 well plate check the Increments box Inc and enter 8 in the Runs field Note The same method and injection time will apply to all runs 10 Select the data directory where the run should be stored Note Subfolders will be created in the data directory by optionally entering User ID and Plate ID in the correspo
18. p fragments Alignment marker with 15 bp and 1 kb fragments Alignment marker with 15 bp and 3 kb fragments Alignment marker with 15 bp and 10 kb fragments Alignment marker with 15 bp and 5 kb fragments Cat no 929550 92955 929552 929553 929555 929556 929557 929558 929520 929521 929522 929523 929524 QlAxcel RNA Handbook 01 2008 Product Contents Cat no QX Alignment Marker Alignment marker with 50 bp and 500 929525 50 bp 500 bp bp fragments 1 5 ml QX Alignment Marker Alignment marker with 50 bp and 1 kb 929526 50 bp 1 kb 1 5 ml fragments QX Alignment Marker Alignment marker with 15 bp and 400 929527 15bp 400bp 1 5 ml bp fragments QX Alignment Marker Alignment marker with 50 bp and 3 kb 929528 50 bp 3 kb 1 5 ml fragments QX Alignment Marker Alignment marker with 50 bp and 5 kb 929529 50 bp 5 kb 1 5 ml fragments QX RNA Alignment RNA alignment marker 929510 Marker 1 5 ml Calibration marker QX Intensity 600 ul QX Intensity Calibration Marker 929500 Calibration Marker 600 ul Buffers QX DNA Dilution 15 ml QX DNA Dilution Buffer 929601 Buffer 15 ml QX RNA Dilution 15 ml QX RNA Dilution Buffer 929602 Buffer 15 ml QX Separation Buffer 40 ml QX Separation Buffer 929603 40 ml QX Wash Buffer 40 ml QX Wash Buffer 929604 40 ml QX Mineral Oil 50 ml QX Mineral Oil 929605 50 ml Accessories QX Cartridge Stand QX Cartridge Stand 929701 QX Buffer T
19. ray QX Buffer Tray 929702 QlAxcel RNA Handbook 01 2008 27 Product Contents Cat no QX 0 2 ml 12 Tube 80 x QX 0 2 ml 12 Tube Strips 929703 Strip 80 QX Colored 0 2 ml 80 x QX Colored 0 2 ml 12 Tube Strips 929704 12 Tube Strip 80 QX Nitrogen Cylinder 6 x QX Nitrogen Cylinder 929705 6 Tubing for external Tubing 10f 3m for external N2 source 9018435 N2 source 28 QlAxcel RNA Handbook 01 2008 www qiagen com Australia Orders 03 9840 9800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 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 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 02 33430411 Fax 02 33430426 Technical 800 787980 Japan Telephone 03 5547 0811 Fax 03 5547 0818 Technical 03 5547 0811 Luxembourg Orders 8002 2076
20. rnight if more convenient before use Autoclaving alone will not fully inactivate many RNases Alternatively glassware can be treated with DEPC diethyl pyrocarbonate Fill glassware with 0 196 DEPC 0 196 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 Solutions Solutions water and other solutions should be treated with 0 196 DEPC DEPC is a strong but not absolute inhibitor of RNases It is commonly used at a concentration of 0 196 to inactivate RNases on glass or plasticware or to create RNase free solutions and water DEPC inactivates RNases by covalent modification Add 0 1 ml DEPC to 100 ml of the solution to be treated and shake vigorously to bring the DEPC into solution Let the solution incubate for 12 hours at 37 C Autoclave for 15 minutes to remove any trace of DEPC DEPC will react with primary amines and cannot be used directly to treat Tris buffers DEPC is highly unstable in the presence of Tris buffers and decomposes rapidly into ethanol and CO When preparing Tris buffers treat water with DEPC first and then dissolve Tris to make the appropriate buffer Trace amounts of DEPC will modify purine residues in RNA by carbethoxylation Carbethoxylated RNA is translated with very low efficiency in cell free systems However its ability to form DNA RNA or RNA RNA hybrids is not seriously affected unless a large fraction of the purine resid
21. s are to be processed the empty wells should be filled with QX RNA Dilution Buffer or a buffer of similar salt concentration to the sample Failure to do this may cause damage to the capillaries For total RNA sample concentration greater than lug ul cRNA concentration greater than 500ng ul or fragmented RNA greater than 500ng ul it is required to dilute the samples to the concentration suggested in Table 1 before performing denaturation Method Selection Preinstalled methods are available for each QlAxcel RNA Gel Cartridge To select the appropriate method for the samples being analyzed refer to Appendix C of the QlAxcel User Manual 14 QlAxcel RNA Handbook 01 2008 Protocol Determining RNA Quality and Quantity Using the QlAxcel RNA Quality Control Kit with the QIAxcel System Important points before starting E Before beginning the procedure read Important Notes beginning on page 11 M For optimal results the solution containing the RNA samples should be approximately pH 7 8 E Determine the optimum QlAxcel method for sample analysis see Appendix C of the QlAxcel User Manual for more details Things to do before starting E Ensure samples have been prepared according to the instructions in Sample preparation recommendations page 13 M Ensure that the QlAxcel Gel Cartridge is set up and all reagents have been prepared according to the instructions in Preparing the QlAxcel Gel Cartridge and buffer tray page 11
22. sable and nondisposable vessels and solutions while working with RNA General handling Proper microbiological aseptic technique should always be used when working with RNA Hands and dust particles may carry bacteria and molds and are the most common sources of RNase contamination Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin or from dusty laboratory equipment Change gloves frequently and keep tubes closed whenever possible Keep purified RNA on ice when aliquots are pipetted for downstream applications 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 Mt a SESESSSaSa _ _ _ _ _ _ _ a s ______ s QIAxcel RNA Handbook 01 2008 23 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 24 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 a detergent thoroughly rinsed and oven baked at 240 C for at least 4 hours ove
23. safety data sheets MSDSs These are available online in convenient and compact PDF format at www giagen com Support MSDS aspx where you can find view and print the MSDS for each QIAGEN kit and kit component 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 AAAARAA DO L C LZEZEZZZAIT QlAxcel RNA Handbook 01 2008 7 Introduction The QlAxcel system when used in conjunction with the QlAxcel RNA Quality Control Kit provides fully avtomated quantitative and qualitative analysis of up to 96 samples per run The QlAxcel RNA Gel Cartridge provides fast and sensitive analysis of the quality and quantity of total RNA single stranded cDNA fragmented or intact or cRNA fragmented or intact Automated sample loading and analysis reduce manual handling of samples minimizing the risk of RNA degradation and contamination The system can detect as little as 5 ng Ll of total RNA and 10 ng Ll of cRNA or single stranded cDNA QlAxcel technology based on capillary electrophoresis using gel cartridges provides unmatched resolution speed and throughput QlAxcel Gel Cartridges are reusable allowing up to 12 x 100 runs to be performed Preinstalled methods suitable for most applications are provid
24. tensity Calibration Marker QX Alignment Marker 12 Tube Strips 929002 929004 929006 929102 Software key allowing use of 9018391 BioCalculator analysis software on an additional computer for data analysis only QIAxcel RNA Handbook 01 2008 25 Product Contents The software key is for analysis of results only It does not provide any instrument control functions DNA size markers QX DNA Size Marker pUC18 Haelll 50 ul QX DNA Size Marker FX174 Haelll 50 pl QX DNA Size Marker 25 bp 1 8 kb 50 ul QX DNA Size Marker 100 bp 3 kb 50 ul QX DNA Size Marker 25 450 bp 50 ul QX DNA Size Marker 50 800 bp 50 ul QX DNA Size Marker 250 bp 4 kb 50 ul QX DNA Size Marker 250 bp 8 kb 50 ul Alignment markers QX Alignment Marker 15 bp 500 bp 1 5 ml QX Alignment Marker 15 bp 1 kb 1 5 ml QX Alignment Marker 15 bp 3 kb 1 5 ml QX Alignment Marker 15 bp 10 kb 1 5 ml QX Alignment Marker 15 bp 5 kb 1 5 ml 26 DNA size marker with 9 fragments 80 589 bp DNA size marker with 11 fragments 72 1353 bp DNA size marker with 12 fragments 25bp 1 8 kb DNA size marker with 14 fragments 100 bp 3 kb DNA size marker with 17 fragments 25 450 bp DNA size marker with 11 fragments 50 800 bp DNA size marker with 11 fragments 250 bp 4 kb DNA size marker with 11 fragments 250 bp 8 kb Alignment marker with 15 bp and 500 b
25. ues have been modified Residual DEPC must always be eliminated from solutions or vessels by autoclaving or heating to 100 C for 15 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 24 QlAxcel RNA Handbook 01 2008 Ordering Information Product QlAxcel Warranty PLUS 2 QlAxcel QIAxcel Kits QIAxcel DNA High Resolution Kit 1200 QlAxcel DNA Screening Kit 2400 QlAxcel DNA Large Fragment Kit 600 QlAxcel RNA Quality Control Kit 1200 Software Data Review Key green Cat no 9001421 Contents Automated system for fast and fully automated DNA fragment analysis or qualitative and quantitative RNA analysis BioCalculator software 1 year warranty on parts and labor 2 or 3 year warranty l preventive 9241202 maintenance visit per year 48 hour 2 working days priority response all labor travel and repair parts QlAxcel DNA High Resolution Gel Cartridge Buffers Mineral Oil QX Intensity Calibration Marker 12 Tube Strips QlAxcel DNA Screening Gel Cartridge Buffers Mineral Oil QX Intensity Calibration Marker 12 Tube Strips QlAxcel DNA Large Fragment Gel Cartridge Buffers Mineral Oil QX Intensity Calibration Marker 12 Tube Strips QlAxcel RNA Quality Control Gel Cartridge Buffers Mineral Oil QX In
26. ve user information is also provided in the Help menu of the BioCalculator software 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 aiagen com 18 QlAxcel RNA Handbook 01 2008 Appendix A Data Analysis Aligning the gel image After the run a gel image window is displayed This is referred to as the Folder View There is an additional window within the Folder View window where data for all channels are visible Data from each individual channel can be opened by double clicking on them All of the data generated are automatically aligned by the BioCalculator software however manual alignment is possible see procedure below Alignment compensates for any slight variations between the different capillaries and results in the alignment of the first and last peak band across all 12 channels Folder View BioCalculator Folder3 File Folder Edit View Analysis Window Help BACHE OSS S55 DHCsMI enl ich iburified PCR A O1 hd 0 X M purified PCR A 04 hd EX AN purified PCR A O7 hd OX 8 8 o its RFU x1E 001 H purified PCR A 01 hda 20 15 164 424 0 455 0 n 99 mm 458 0 4340 298 0 298 0 2870 257 257 0 287 0 o 1740 174 0 Rel Units RU x 1 E1000
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