June QIAxcel RNA Handbook. For automated quantitative and qualitative RNA analysis using the QIAxcel system. Sample & Assay Technologies

Transcription

1 June 2015 QIAxcel RNA Handbook For automated quantitative and qualitative RNA analysis using the QIAxcel system Sample & Assay Technologies

2 QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies, enabling the isolation and detection of contents of any biological sample. Our advanced, high-quality products and services ensure success from sample to result. QIAGEN sets standards in: Purification of DNA, RNA, and proteins Nucleic acid and protein assays microrna research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs. For more information, visit

3 Contents Kit Contents 5 Unpacking the QIAxcel Gel Cartridge 5 Storage 5 Intended Use 6 Product Warranty and Satisfaction Guarantee 7 Technical Assistance 7 Quality Control 7 Safety Information 8 Introduction 9 Principle and procedure 9 Equipment and Reagents to Be Supplied by User 11 Important Notes 12 Preparing the QIAxcel RNA gel cartridge and buffer tray 12 Sample preparation recommendations 17 Method selection 18 Protocols Determination of RNA Quality and Quantity using QIAxcel ScreenGel Software 19 Determination of RNA Quality and Quantity using BioCalculator Software 22 Troubleshooting Guide 26 Appendix A: Creating a Process Profile in QIAxcel ScreenGel Software 29 Appendix B: Data Analysis 30 Data analysis with the QIAxcel ScreenGel Software 30 Data analysis with BioCalculator Software 39 Appendix C: General Remarks on Handling RNA 43 Appendix D: Removing Gel from Blocked Capillaries 45 Standard gel-droplet test 45 Gel-droplet test with hot water 47 Gel-droplet test using the QX Cartridge Prep Station 48 Performing a signal check 49 QIAxcel RNA Handbook 06/2015 3

4 References 51 Ordering Information 52 4 QIAxcel RNA Handbook 06/2015

5 Kit Contents QIAxcel RNA QC Kit v2.0 (1200) Catalog no Number of assays 12 x 100 QIAxcel RNA Quality Control Cartridge (with smart key) 1 QX Separation Buffer* QX Wash Buffer* QX Mineral Oil QX RNA Dilution Buffer 100 ml 40 ml 50 ml 15 ml QX Intensity Calibration Marker 600 µl QX 0.2 ml 12-Tube Strips 2 QX Colored 0.2 ml 12-Tube Strips 2 QX RNA Alignment Marker 1.5 ml QX RNA Size Marker nt 2 x 20 µl QX RNA Denaturation Buffer 2 x 2 ml Handbook 1 * Contains sodium azide as a preservative. Unpacking the QIAxcel Gel Cartridge For optimal performance, store the QIAxcel RNA gel cartridge at 2 8 C until the first use. Prior to use, the QIAxcel RNA gel cartridge should be placed into the QX Cartridge Stand and protected with the cover whenever exposed to light, or stored latched in the instrument in the Park Position with buffer in the buffer tray, and allowed to stand for at least 20 minutes (for more information, see Preparing the QIAxcel RNA gel cartridge and buffer tray, page 12). Storage Note: QX RNA Denaturation Buffer and QX RNA Size Marker nt are delivered separately. QX RNA Size Marker nt should be stored at 20 to 80 C, and is stable for at least 6 months under these conditions. QIAxcel RNA Handbook 06/2015 5

6 QX RNA Denaturation Buffer should be stored at 2 8 C and is stable for at least 6 months under these conditions. All other components of the QIAxcel RNA QC Kit v2.0, except for the QIAxcel RNA gel cartridge, can be stored dry at room temperature (15 25 C) until the expiration date as indicated on the label. Store QIAxcel RNA gel cartridges at 2 8 C until the first use. If the QIAxcel RNA gel cartridge is not being used on a daily basis, close the purge port with the purge port seal, return the QIAxcel RNA gel cartridge to the blister package, inserting the capillary tips into the soft gel, and store at 2 8 C in an upright position (see orientation label on blister package). Note: Storing the QIAxcel RNA gel cartridge below 2 C can severely damage the cartridge. Prior to use, the QIAxcel RNA gel cartridge should be placed into the QX Cartridge Stand and protected with the cover whenever exposed to light, or stored latched in the instrument in the Park Position with buffer in the buffer tray, and allowed to stand for at least 20 minutes. If the QIAxcel RNA gel cartridge is being used on a daily basis, store the cartridge latched in the QIAxcel instrument in the Park Position (see Section 5.3 of the QIAxcel User Manual or the QIAxcel Advanced User Manual). Note: The QIAxcel instrument must be left on if the cartridge is stored in the Park Position and the cartridge must be latched. Do not switch the QIAxcel instrument off. If more than one QIAxcel RNA gel cartridge is being used on a daily basis, store the second cartridge in the QX Cartridge Stand in the dark or protected with the cover. Make sure that the cartridge stand reservoir is filled with wash buffer and covered with mineral oil (see Section 5.1 of the QIAxcel User Manual or Section of the QIAxcel Advanced User Manual). Alternatively, close the purge port with the purge port seal, return the QIAxcel RNA gel cartridge to the blister package, inserting the capillary tips into the soft gel, and store at 2 8 C in an upright position (see orientation label on blister package). The QIAxcel RNA QC Kit v2.0 can be stored in this manner until expiration date as indicated on the kit label. Intended Use The QIAxcel RNA QC Kit v2.0 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 QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments, or to other applicable guidelines. 6 QIAxcel RNA Handbook 06/2015

7 Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature. The purchaser must determine the suitability of the product for their 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 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 QIAxcel RNA QC Kit v2.0 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 or call one of the QIAGEN Technical Service Departments or local distributors (see back cover or visit Quality Control In accordance with QIAGEN s ISO-certified Quality Management System, each lot of QIAxcel RNA QC Kit v2.0 is tested against predetermined specifications to ensure consistent product quality. QIAxcel RNA Handbook 06/2015 7

8 Safety Information When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate safety data sheets (SDSs). These are available online in convenient and compact PDF format at where you can find, view, and print the SDS for each QIAGEN kit and kit component. 8 QIAxcel RNA Handbook 06/2015

9 Introduction QIAxcel instruments, in combination with the QIAxcel RNA QC Kit v2.0, provide fully automated quantitative and qualitative analysis of up to 96 RNA samples per run. The QIAxcel RNA Quality Control 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/µl of total RNA and 10 ng/µl of crna or single-stranded cdna in a final volume of 10 µl. QIAxcel technology, based on capillary electrophoresis using QIAxcel gel cartridges, provides unmatched resolution, speed, and throughput. The QIAxcel RNA Quality Control Cartridge is reusable, allowing up to 100 runs of 12 samples to be performed. QIAxcel instruments are preinstalled with methods suitable for most applications. In addition, customized methods can also be created contact QIAGEN Technical Services for more details. QIAxcel ScreenGel Software, supplied with the QIAxcel Advanced instrument, provides both electropherogram and gel images of nucleic acid separation. The QIAxcel DNA High Resolution Kit, the QIAxcel DNA Screening Kit, and the QIAxcel Fast Analysis Kit can also be used with the QIAxcel system (see ordering information, page 53). These kits allow fast qualitative and quantitative analysis of DNA fragments. The QIAxcel Protein Kit for protein separation and quantification is also available (see ordering information, page 53). Principle and procedure The QIAxcel system uses capillary gel electrophoresis to enable fast separation of nucleic acids based on size. Unlike traditional agarose gel electrophoresis, separation is performed in a capillary of a precast gel cartridge. Each sample is automatically loaded into an individual capillary (according to voltage and time parameters) and voltage is applied. The negatively charged nucleic acid molecules migrate through the capillary to the positively charged end (Figure 1, page 10). As with agarose gel electrophoresis, low-molecular-weight molecules migrate faster than high-molecular-weight molecules. As the molecules migrate through the capillary, they pass a detector which detects and measures the fluorescent signal. A photomultiplier detector converts the emission signal into electronic data, which are then transferred to the computer for further processing using QIAxcel ScreenGel Software or BioCalculator Software. After processing, the data are displayed as an electropherogram or gel image. QIAxcel RNA Handbook 06/2015 9

10 The QIAxcel system offers a number of advantages over traditional agarose gel electrophoresis, including: Higher detection sensitivity Less sample wastage (minimal sample input volumes) Fast analysis of up to 96 samples Automated loading and analysis Figure 1. Sample separation process using the QIAxcel system. Nucleic acid molecules are separated according to size by applying an electrical current to a gel-filled capillary. A photomultiplier detector in the instrument detects the nucleic acid molecules as they migrate towards the positively charged end of the capillary. The data are converted to an electropherogram and a gel image by the QIAxcel ScreenGel Software or the BioCalculator Software (not shown). (Note: data shown are for DNA; similar data are obtained for RNA.) 10 QIAxcel RNA Handbook 06/2015

11 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 (SDSs), available from the product supplier. For all users Pipets and pipet tips 12-tube strips (e.g., QX 0.2 ml 12-Tube Strip, cat. no ) or 96-well plates Centrifuge with rotor suitable for 0.2 ml strips or 96-well plates, such as the Centrifuge 4-16 or Centrifuge 4-16K (for ordering information, see RNase-free water PCR cycler, heat block, or water bath for denaturing RNA samples For QIAxcel users QIAxcel instrument (cat. no ) and BioCalculator Software or QIAxcel ScreenGel Software For QIAxcel Advanced users QIAxcel Advanced instrument (cat. no ) and QIAxcel ScreenGel Software Note: If working with RNA for the first time, read Appendix C (page 43). QIAxcel RNA Handbook 06/

12 Important Notes Preparing the QIAxcel RNA gel cartridge and buffer tray This procedure describes how to prepare the QIAxcel RNA Quality Control Cartridge and buffer tray prior to RNA analysis. Important points before starting The volume of buffer supplied is sufficient for a defined number of sample runs. If required, additional buffers can be purchased separately (see ordering information, page 53). The 0.2 ml 12-tube strips containing QX RNA Alignment Marker and QX Intensity Calibration Marker (if required) should fit loosely in the MARKER1 and MARKER2 positions (see Preparing QX RNA Alignment Marker, page 14, and Intensity calibration, page 15). QX RNA Alignment Marker should be replaced every runs or 3 days, whichever comes first. Additional markers and buffers may need to be purchased (see ordering information, page 53). When not in use, the 12-tube strip containing QX RNA Alignment Marker should be stored at 20 C. For optimal performance, store the QIAxcel RNA gel cartridge at 2 8 C until required for use. Prior to use, the QIAxcel RNA gel cartridge should be placed into the QX Cartridge Stand and protected with the cover, or stored latched in the instrument in the Park Position with buffer in the buffer tray, and allowed to stand for at least 20 minutes. Things to do before starting If prepared in advance, the 12-tube strip containing QX RNA Alignment Marker should be equilibrated to room temperature (15 25ºC) and centrifuged briefly before use. If the QIAxcel RNA gel cartridge is being used for the first time, intensity calibration should be performed (see Intensity calibration, page 15 and Section 5.4 of the QIAxcel User Manual or Section of the QIAxcel Advanced User Manual). This is not necessary if the QIAxcel RNA gel cartridge has already been calibrated, unless it is being used on a different QIAxcel or QIAxcel Advanced instrument or a different computer is used to operate the instrument. If a different computer is being used to operate the QIAxcel or QIAxcel Advanced instrument, the calibration log file must be transferred to the new computer so that calibration does not need to be performed again. 12 QIAxcel RNA Handbook 06/2015

13 Unpacking and preparing the QIAxcel RNA gel cartridge For optimal performance, store the QIAxcel RNA gel cartridge at 2 8 C until required for use. Prior to use, the QIAxcel RNA gel cartridge should be placed into the QX Cartridge Stand protected with the cover, or stored latched in the instrument in the Park Position with buffer in the buffer tray, and allowed to stand for at least 20 minutes. 1. Remove all buffer bottles from the kit box. 2. Add 10 ml QX Wash Buffer to both reservoirs of the QX Cartridge Stand (provided with QIAxcel instruments) and cover with 2 ml mineral oil (supplied). 3. Remove the QIAxcel RNA gel cartridge from its packaging and carefully wipe off any soft gel debris from the capillary tips using a soft tissue. 4. Remove the purge cap seal from the back of the QIAxcel RNA gel cartridge and place the gel cartridge in the QX Cartridge Stand. Retain the purge port seal in case you need to store the QIAxcel RNA gel cartridge. Note: Use a soft tissue to wipe off any gel that may have leaked from the purge port. Note: Ensure that the capillary tips are submerged in QX Wash Buffer. 5. Incubate new cartridges in the QX Cartridge Stand for at least 20 minutes prior to use. Note: Once used, the QIAxcel RNA gel cartridge must be stored in a vertical orientation. For more information, see Storage, page 5. Preparing the QIAxcel RNA gel cartridge. QIAxcel RNA Handbook 06/

14 Preparing the buffer tray 1. Allow all reagents to equilibrate to room temperature (15 25 C) before use. 2. Wash the buffer tray with hot water and rinse thoroughly with deionized water. 3. Fill the WP and WI positions of the buffer tray with 8 ml QX Wash Buffer. 4. Fill the BUFFER position of the buffer tray with 18 ml QX Separation Buffer. 5. Carefully 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 mineral oil to position BUFFER. 6. Insert the buffer tray into the buffer tray holder so that the slots for the 12-tube strips face the front of the instrument. Preparing QX RNA Alignment Marker 1. Load 15 µl QX RNA Alignment Marker into each tube of a QX 0.2 ml 12-Tube Strip. 2. Add 1 drop of mineral oil to each tube, and place the strip into the MARKER1 position of the buffer tray. Preparing the buffer tray and inserting the buffer tray into the buffer tray holder. Installing the QIAxcel RNA gel cartridge and smart key 1. Remove the QIAxcel RNA gel cartridge from the QX Cartridge Stand. 2. Open the cartridge door and place the QIAxcel RNA gel cartridge into the QIAxcel or QIAxcel Advanced instrument. The cartridge description label should face the front and the purge port should face the back of the instrument. 3. Insert the smart key into the smart key socket. The smart key can be inserted in either direction. 4. Close the cartridge door. 14 QIAxcel RNA Handbook 06/2015

15 5. The cartridge identifier, number of runs remaining, and cartridge type will be displayed automatically in the software when the cartridge smart key is inserted. Note: The system will not recognize the cartridge and will not operate if the smart key is not inserted. A B Installing the QIAxcel gel cartridge and smart key in the A QIAxcel and B QIAxcel Advanced instruments. Intensity calibration Every QIAxcel gel cartridge requires intensity calibration prior to sample analysis. The intensities of each capillary are normalized and a factor is applied for every subsequent run. This corrects for natural intensity reading variations between each capillary in the cartridge. Intensity calibration using QIAxcel ScreenGel Software The data for each cartridge intensity calibration are stored in a file named according to the cartridge and instrument identifiers (<cartridgeid>_<instrument-id>.xcc). This file is saved either in the default directory C:\Documents and Settings\All Users\Application Data\QIAGEN\QIAxcel\ScreenGel\Data\CartridgeCalibrationData or in a customized directory. If, for any reason, a different computer is used to the one on which the calibration file is saved, the calibration file should be transferred to the new computer. Otherwise, recalibration of the cartridge is required. Similarly, if the QIAxcel gel cartridge is used on a different QIAxcel instrument to the one it was calibrated on, another intensity calibration should be performed. QIAxcel RNA Handbook 06/

16 Intensity calibration of the cartridge takes about 16 minutes. 1. Load 15 µl QX Intensity Calibration Marker into each tube of a QX Colored 0.2 ml 12-Tube Strip. Add a drop of mineral oil, and insert the strip into the MARKER2 position of the buffer tray. 2. Launch the calibration run by clicking the Start calibration button in the Calibration screen of the Service environment. 3. Once the calibration is complete, the calibration results are displayed next to the gel image or the electropherogram view. The result table shows the area, calibration factor, and the result ( Pass or Fail ) for each channel. Note: A successfully calibrated cartridge should have a normalized area calibrated range between If one or more channels show no signals in the first run, see Appendix D, page If one or more channels show high background, see Section 8 of the QIAxcel User Manual or the QIAxcel Advanced User Manual. 6. If calibration fails more than twice, call QIAGEN Technical Services. Recalibration using QIAxcel ScreenGel Software To recalibrate a cartridge, repeat the procedure described in Intensity calibration using QIAxcel ScreenGel Software, page 15. The calibration results of the previous calibration procedure are discarded when recalibrating a cartridge. Note: It is possible to calibrate a cartridge for which no calibration runs remain. In this case, 3 of the remaining regular runs are used instead of 1 calibration run. Intensity calibration using BioCalculator Software The data (individual calibration data files) for each cartridge intensity calibration are stored in the CALdata folder. This folder is saved in the BioCalculator root directory: C:\Program Files\QIAxcel BioCalculator. A Calibration2.log file (cartridge calibration information) is saved automatically in the BioCalculator root directory: C:\Program Files\QIAxcel BioCalculator. If, for any reason, a different computer is used to the one on which the Calibration2.log file is saved, the Calibration2.log file should be transferred to the new computer. Otherwise, recalibration of the cartridge is required. Similarly, if the QIAxcel gel cartridge is used on a different QIAxcel instrument to the one it was calibrated on, another intensity calibration should be performed. 16 QIAxcel RNA Handbook 06/2015

17 Intensity calibration of the cartridge takes about 16 minutes. 1. Load 15 µl QX Intensity Calibration Marker into each tube of a QX Colored 0.2 ml 12-Tube Strip, make sure no air bubbles are trapped in the solution, and place it into the MARKER2 position of the buffer tray. 2. Launch the calibration wizard by selecting File then Intensity Calibration in the Instrument Control window. 3. Click Start to begin the cartridge intensity calibration. 4. When calibration is complete, the Calibration Verification dialog box will open. This will show either Pass or Fail for each channel. Note: A successfully calibrated cartridge should have a normalized area calibrated range between If one or more channels fail, repeat the calibration process using a new strip of QX Intensity Calibration Marker (see Recalibration process, page 17). 6. If one or more channels show no signals in the first run, see Appendix D, page If one or more channels show high background, see Section 8 of the QIAxcel User Manual or the QIAxcel Advanced User Manual. 8. If calibration fails more than twice, call QIAGEN Technical Services. Recalibration using BioCalculator Software Note: Use a new strip of QX Intensity Calibration Marker for each recalibration run. Re-using the intensity calibration marker may affect the calibration data and can cause them to be out of range. 1. Load 15 µl QX Intensity Calibration Marker into each tube of a new QX Color 0.2 ml 12-Tube Strip. Make sure there are no air bubbles, and place the strip into the MARKER2 position of the buffer tray. 2. Launch the calibration wizard by selecting File then Intensity Calibration in the Instrument Control window. 3. Click Recalibrate and then Start to repeat the calibration routine. Sample preparation recommendations The minimum sample volume required for analysis is 10 µl. Less than 0.1 µl of the sample will be injected into the QIAxcel RNA gel cartridge for analysis. Typical sample preparation procedure 1. Dilute RNA in nuclease-free water as described in Table 1 (next page). Note: QX RNA Size Marker nt is used without dilution. QIAxcel RNA Handbook 06/

18 Table 1. Suggested RNA concentrations RNA sample type Suggested concentration (ng/µl) Total RNA Method CM-RNA CL-RNA crna or singlestranded RNA CM-RNA CL-RNA Fragmented crna or cdna CM-F-RNA 2. Pipet RNA samples (1 µl total, 1 µg/µl) and QX RNA Size Marker nt (1 µl) into the tubes of a 0.2 ml 12-Tube Strip. 3. Add an equal volume of QX RNA Denaturation Buffer. 4. Heat the solution at 70 C for 2 minutes on a heating block or in a PCR machine, then place on ice for 1 minute. 5. Centrifuge briefly to collect any condensation. 6. Bring the total sample volume to 10 µl 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 samples 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 a total RNA sample concentration greater than 1 µg/µl, crna concentration greater than 500 ng/µl, or a fragmented RNA concentration greater than 500 ng/µl, dilute the samples to the concentration suggested in Table 1 before performing denaturation. For more dilute RNA samples, 2 µl of RNA and denaturation buffer may be used. Use of larger volumes may lead to abnormal migration and signal intensities. Use of significantly longer injection times (beyond 20 seconds) may lead to peak broadening and is therefore not recommended. Method selection A number of preinstalled methods are available for QIAxcel gel cartridges. To select the appropriate method for the samples being analyzed, see Table 1 (page 18), Section 5.5 of the QIAxcel User Manual, or Section of the QIAxcel Advanced User Manual. 18 QIAxcel RNA Handbook 06/2015

19 Protocol: Determination of RNA Quality and Quantity using QIAxcel ScreenGel Software Important points before starting Before beginning the procedure, read Important Notes beginning on page 12. For optimal results, RNA should be in a solution of approximately ph 7 8. Determine the optimal QIAxcel method for sample analysis (see Table 1, page 18, Section 5.5 of the QIAxcel User Manual, or Section of the QIAxcel Advanced User Manual for more details). Start the software in RNA mode. To switch to RNA mode, log out of the software and log in again in the RNA mode (for more information, see Appendix B, page 30). The steps in this protocol are based on the default RNA QC profile while logged in as a routine user. Position A12 is assigned as the size marker. Things to do before starting Ensure samples have been prepared according to the instructions in Sample preparation recommendations, page 17. If the QIAxcel RNA gel cartridge was stored at 2 8 C, place it into the QX Cartridge Stand or the instrument, latched in the Park Position with buffer in the buffer tray, and allow to stand for at least 20 minutes prior to use. Ensure that the QIAxcel RNA gel cartridge is correctly set up and all reagents have been prepared according to the instructions in Preparing the QIAxcel RNA gel cartridge and buffer tray, page Switch on the QIAxcel instrument. 2. Switch on the computer and launch the QIAxcel ScreenGel Software. 3. Log in in the RNA mode. To switch to RNA mode, log out of the software and log in again in the RNA mode. 4. Install the QIAxcel RNA gel cartridge. See Section of the QIAxcel User Manual or the QIAxcel Advanced User Manual for more details. QIAxcel RNA Handbook 06/

20 5. Load the buffer tray containing the QX RNA Alignment Marker into the buffer tray holder. See Section of the QIAxcel User Manual or the QIAxcel Advanced User Manual for more details. Note: If being used for the first time, the QIAxcel RNA gel cartridge will require calibration (see Intensity calibration using QIAxcel ScreenGel Software page 15). Note: QX RNA Alignment Markers should be replaced every runs or 3 days, whichever comes first. When not in use, the 12-tube strip containing QX RNA Alignment Marker should be stored at 20 C. 6. Load the sample strips or load a 96-well plate containing samples onto the sample tray holder. Note: The cartridge door and sample door of the QIAxcel instrument 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. 7. Select a process profile from the drop-down list. Note: Process profiles provide preset analysis and report parameters for a single row of samples. Default process files or user-created files can be used. See Appendix A, page 29 or Section 6.3 of the QIAxcel Advanced User Manual for a description of how to create process profiles. 8. Click Next to open the Sample Selection tab. The following information can be modified in this tab: size and alignment marker selection and position, lot number information, and the automatically generated plate identifier. 20 QIAxcel RNA Handbook 06/2015

21 9. Click Next to open the Sample Information tab. Information about the sample can be entered. 10. Click Next to open the Run Check tab, and confirm that samples and markers have been loaded correctly. 11. Click Run to start the run. Note: A report is automatically generated according to the settings in the selected process profile. Note: Changes to the analysis settings can be performed by an advanced user. QIAxcel RNA Handbook 06/

22 Protocol: Determination of RNA Quality and Quantity using BioCalculator Software Important points before starting Before beginning the procedure, read Important Notes beginning on page 12. For optimal results, RNA should be in a solution of approximately ph 7 8. Determine the optimal QIAxcel method for sample analysis (see Table 1, page 18, or Section 5.5 of the QIAxcel User Manual for more details). RNA methods can be run on the QIAxcel instrument using BioCalculator Software in normal or RNA mode. For analysis of RNA data, including automatic analysis after the run (see step 14, page 24), the software must operate in RNA mode. To switch to RNA mode, close the Instrument Control window and any data files that are open (for more information, see Appendix B, page 30). Things to do before starting Ensure samples have been prepared according to the instructions in Sample preparation recommendations, page 17. If the QIAxcel RNA gel cartridge was stored at 2 8 C, place it into the QX Cartridge Stand or the instrument latched in the Park Position with buffer in the buffer tray, and allow to stand for at least 20 minutes prior to use. Ensure that the QIAxcel RNA gel cartridge is correctly set up and all reagents have been prepared according to the instructions in Preparing the QIAxcel RNA gel cartridge and buffer tray, page 12. Procedure 1. Switch on the QIAxcel instrument. 2. Switch on the computer and launch the BioCalculator Software. 3. Optional: Switch to the RNA mode. 4. Install the QIAxcel RNA gel cartridge. See Section of the QIAxcel User Manual for more details. 22 QIAxcel RNA Handbook 06/2015

23 5. Load the buffer tray containing the QX RNA Alignment Marker into the buffer tray holder. See Section of the QIAxcel User Manual for more details. Note: If being used for the first time, the QIAxcel RNA gel cartridge will require calibration (see Intensity calibration using BioCalculator Software page 16). Note: QX RNA Alignment Markers should be replaced every runs or 3 days, whichever comes first. When not in use, the 12-tube strip containing QX RNA Alignment Marker should be stored at 20 C. 6. Load the sample strips or load a 96-well plate containing samples onto the sample tray holder. Note: The cartridge door and sample door of the QIAxcel 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. 7. Select the appropriate method in the Instrument Control window. See Table 1 (page 18) or Section 5.5 of the QIAxcel User Manual for more information about preinstalled methods. QIAxcel RNA Handbook 06/

24 8. Enter the sample name, position, and number of runs in the corresponding fields of the Instrument Control window. 9. Optional: Enter the sample injection time (minimum: 5 s; maximum: 40 s) into the Time field. If the Time field is left blank, the QIAxcel system uses the default settings for the selected method. 10. To perform multiple analyses of the same row, enter the number of repeats in the Runs field and leave the increments box ( Inc ) unchecked. To process all rows of a 96-well plate using the same method, check the increments box ( Inc ) and enter 8 in the Runs field. 11. Select the data directory where the run should be saved. Note: Subfolders will be created in the data directory if information is entered into the User ID and Plate ID fields. Optional: If connected to a network, in addition to a local data directory, you are able to select a network directory in which the run data will be saved. 12. Recommended: Click the Sample Info button to enter sample information for each well. Alternatively, sample information in a spreadsheet can be imported in *.csv (comma-separated value) file format. 13. Make sure to check the separation channels to be used (i.e., if running less than the maximum number of samples, check only the channels that contain samples). Note: Unused wells should contain QX RNA Dilution Buffer to prevent damage to the channel. 14. Optional: Check the Automatically analyze after data acquisition box, check the Include reference marker table box, click the Marker button, and open the desired RNA reference marker table. When this option is checked, analysis will be performed using reference marker data from a preexisting reference marker table. Note: The software must be operating in RNA mode (see Appendix B, page 30) to allow automatic RNA analysis after data acquisition. 15. Check the status of the QIAxcel system in the status panel. Make sure that the cartridge door and sample door are closed. Note: The status panel is at the bottom of the Instrument Control window and displays information about the status of the QIAxcel system (see Section 5.3 of the QIAxcel User Manual for more details). 24 QIAxcel RNA Handbook 06/2015

25 16. Click Run to start sample processing. At the start of the run, a window displaying electropherogram and gel images opens. QIAxcel RNA Handbook 06/

26 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise. In addition, extensive user information is also provided in the QIAxcel User Manual, the QIAxcel Advanced User Manual and the Help menu of QIAxcel ScreenGel Software and BioCalculator Software. For more information, see also the Frequently Asked Questions page at our Technical Support Center: 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 Incorrect fragment size a) Data in reference marker table is not valid b) Artifacts in reference marker channel c) Reference marker or RNA sample not denatured Comments and suggestions Make sure the correct lane is selected as size marker and all fragments are correctly identified (QIAxcel ScreenGel Software). Make sure that the reference marker channel is the active window when using the Fill command to update migration time and peak area data in the marker table (BioCalculator Software). Make sure that the reference marker table contains data for all 8 fragments. If not, reload the marker table from the file or use the default reference marker table. Occasionally, artifacts in the reference marker channel can interfere with correct peak identification. In this case, use a reference marker table from another run or repeat the run. Artifact peaks can be removed manually using the QIAxcel ScreenGel Software. Make sure to denature all samples according to the recommendations in Intensity calibration using BioCalculator Software, page QIAxcel RNA Handbook 06/2015

27 d) Reference marker data not current Comments and suggestions If the data in the reference marker table are not up to date (e.g., a different cartridge was used or the cartridge was used for more than 5 10 runs), migration times may deviate from those in the current run. Include fresh reference marker at least every 5 10 runs when reusing a QIAxcel gel cartridge and each time a new QIAxcel gel cartridge is used for the first time. Ribosomal RNA peaks not identified correctly Settings in reference marker table incorrect Estimated RNA concentration is incorrect a) Cartridge not calibrated Manually identify the alignment marker, 18S and 28S peaks, as described in Appendix B, page 30. Make sure to calibrate each cartridge as described in Intensity calibration, page 15. b) Salt content in samples Salt content of samples affects injection efficiency and RNA migration. c) Amount of RNA size marker is incorrect RNA pellets isolated by precipitation-based protocols require careful washing or additional cleanup using spin columns (e.g., RNeasy MinElute Cleanup Kit, cat. no ). If the RNA size marker is partially degraded or the signal is weaker than usual, this will lead to overestimation of RNA concentration. Use a fresh aliquot of RNA size marker. If more than 1 µl of RNA is loaded, the concentration should be corrected accordingly. One or more channels do not appear in the results table a) Wrong channel selected for size marker When using the BioCalculator Software, make sure that the channel containing the size marker is correctly selected from the drop-down list of the reference table (see Appendix B, page 41). QIAxcel RNA Handbook 06/

28 b) Size markers in different positions when several runs analyzed in parallel Comments and suggestions When using QIAxcel ScreenGel Software, the size marker lanes for each batch is selected in the process profile. When using BioCalculator Software, the size marker selected from the drop-down list in the reference marker table is omitted from the results table in all runs included in the analysis. To include all channels, select N/A from the dropdown list. In the full results table, disregard the size marker row (size marker fragments may be erroneously recognized as rrna fragments). 28 QIAxcel RNA Handbook 06/2015

29 Appendix A: Creating a Process Profile in QIAxcel ScreenGel Software Note: Only users with the assigned role Advanced User can create a new process profile. To create a new process profile, proceed as follows: A1. Open the Process environment. If not open, switch to the Process environment by clicking the Process environment icon. Select the Process Profile screen. Main steps for creation of a new process profile. Note: If the last process only just finished, click Back to Wizard at the bottom right. A2. Select the RNA cartridge type from the drop-down list. Note: If the instrument is connected, the system automatically detects the inserted cartridge. A3. Select a process profile. Use the Process Profile drop-down list to select a process profile. The selected profile serves as a template for the creation of the new profile. Note: Select NewProcessProfile to create a process profile from scratch. The system will show *NewProcessProfile after selection. This can only be carried out by users with the assigned role Advanced User. A4. Set the process profile options according to your needs. QIAxcel RNA Handbook 06/

30 See Section of the QIAxcel Advanced User Manual for detailed information. A5. Save the modified process profile under a new name. Click the Save Process Profile as... button below process setup and enter a new unique profile name in the Save Profile dialog box that appears. Note: This can only be carried out by users with the assigned user role Advanced User. Note: If there are process profile screens containing incomplete or inconsistent data highlighted in yellow, the Save process profile as... button is disabled. Select the marked process profile screens and correct the data. If all data are correct, the Save process profile as... button becomes enabled and the process profile can be saved. Appendix B: Data Analysis Data analysis with the QIAxcel ScreenGel Software If the analysis option in the process profile was enabled, data are automatically analyzed and aligned by the QIAxcel ScreenGel Software. Manual analysis can be performed when logged in as an Advanced or Basic user. Before starting the analysis, make sure the QIAxcel ScreenGel Software is in RNA mode by saving your changes, logging out, and logging in again in the RNA mode. We recommend including the QX RNA Size Marker nt in at least 1 capillary when a gel cartridge is used for the first time. Size marker data can be used in subsequent runs; however, size and concentration calculations are more accurate when markers are included in each run. After the run, the gel image is displayed in the analysis environment. Data for each individual channel can be displayed by selecting and changing to the electropherogram view. The analysis of RNA samples is performed in a single step. After the peak detection in the raw data, the peak positions and peak concentrations are determined by mapping the sample to a reference marker. Procedure B1. Load the samples to analyze using the Experiment Explorer. B2. If a size marker was included in the run, create a new reference marker as described in Creating a RNA reference marker in QIAxcel ScreenGel Software, page QIAxcel RNA Handbook 06/2015

31 B3. Select the samples to analyze in the Gel Image view. B4. Open the Analysis tab and select the reference marker table to be used for sample analysis. Note: Newly created reference markers are preselected. In Reference marker, select Reference Marker Table and select a predefined reference marker. Reference marker details are displayed in the marker table. Note: If a reference marker table is not visible, you can display one using the View menu (selecting the menu item View / Show Analysis Parameters ) or by clicking the icon at the extreme right of the view selection bar. Note: To ensure compatibility, the Reference Marker drop-down list is enabled only when a reference marker table matching the alignment marker, cartridge used, and run method of the samples is available. When no matching reference marker is available, a new reference marker can be created (see Creating a RNA reference marker in QIAxcel ScreenGel Software, page 37). Note: For ScreenGel Software version 1.0.x only: In RNA mode, analysis properties affect only the analysis of size marker samples for creating reference markers. All other samples are analyzed using a special RNA algorithm with fixed parameters. B5. In the gel image overview, select samples. QIAxcel RNA Handbook 06/

32 B6. Start the analysis by clicking the Start Analysis button. Analysis results are shown in the result table and in the Single Electropherogram View. B7. Check that fragments were detected for all analyzed samples by inspecting the result table below the gel image or the electropherogram overview. Ensure that the peak detection has worked as expected. If the first detected peak is not the alignment marker peak, as a first step, increase the alignment marker threshold. If this is not sufficient, add the parameter "Suspend Integration" and use the "Absolute" time unit to define the starting point as 0 minutes. Set the ending point of this parameter to shortly before the start of the alignment marker peak (e.g., set end to 2 minutes if the first alignment marker peak appears at 2.5 minutes). If the analysis profile is too sensitive and detects peaks where there is only noise, increase the threshold (e.g., from 2 S/N to 5 S/N). If the baseline follows the 28S peak, increase the baseline filter window size for roughly the last third of the migration time. This is done by clicking the "Add" button to add a second interval to the baseline filter parameter (e.g., set start to 6 minutes and the window size to 80 seconds). Click the "Start Analysis" button again to start a new analysis according to this modified analysis profile. In some cases, it may be necessary to repeat the adaptations to the analysis profile iteratively. If the peak detection worked as expected with the modified analysis profile, save this analysis profile and try to use this new profile instead of the "Default RNA" analysis profile for future RNA analyses. Note: For quality assessment using the "RNA Integrity Score (RIS)" or ratio calculation based on peak calling, proceed with RNA quality assessment using QIAxcel ScreenGel Software, below. B8. Save the analysis results by clicking the Save button in the Experiment Explorer. 32 QIAxcel RNA Handbook 06/2015

33 RNA quality assessment using QIAxcel ScreenGel Software ScreenGel Software 1.2 provides a "RNA Integrity Score (RIS)" function for eukaryotic RNA. For more details on the RNA Integrity Score, refer to the QIAxcel Advanced User Manual. The peaks for RNA quality assessment (RIS or optional 18S:28S ratio) are detected using the estimated (relative) time values and tolerances defined in the peak calling. When some peaks are not identified or size calculation is not possible, the RIS and 18S:28S ratio cannot be calculated. Performing quality control of RNA, with a size marker B1. Analyze the samples using a reference marker. For details, refer to Data analysis with the QIAxcel ScreenGel Software, page 30. B2. Switch to the "Peak Calling" tab of the parameters panel and select one of the provided peak calling instructions: "Default RNA QC", "Default RNA procaryote", or "Default RNA rat_mouse_human". B3. To determine the RNA integrity, select the "RNA Integrity Score" option and choose the 18S peak. To examine the 28S-to-18S-ratio in one sample, select the "Ratio Normalized Area" option and choose the 28S and the 18S peak; for prokaryotic RNA, choose the 23S and 16S peak, respectively. For detailed information on how to define peak calling instructions, refer to the section Modifying peak calling instructions in the QIAxcel Advanced User Manual. QIAxcel RNA Handbook 06/

34 Peak calling instruction for RNA QC. B4. In the gel image overview, select all samples. B5. Click the "Start Peak Calling" button. 34 QIAxcel RNA Handbook 06/2015

35 B6. Inspect the peak calling result in the peak calling result overview. Each row in this table represents the peak calling result for one sample. The RNA integrity score is given in the column "RIS". The 28S-to-18S-ratio, is given in the column "Ratio". In addition, the total concentration of RNA is given in the column "Total RNA concentration". Furthermore, for the 18S and 28S peak, it is listed whether or not this peak was found, based on the criterion given in the peak calling instruction. For detailed information on the peak calling result table, refer to the section Peak Calling in the QIAxcel Advanced User Manual. Performing quality control of RNA, without a size marker B1. Analyze the samples either with no marker or as described above. B2. Switch to the "Peak Calling" tab of the parameters panel and select one of the peak calling instructions: "Default RNA QC", "Default RNA procaryote", or "Default RNA rat_mouse_human". B3. Adapt the peak calling instruction to search for peaks based on relative migration time instead of size. Click on the 18S peak and update the position criterion. Set the value to the calculated relative migration time that is shown in the result table for this peak, change the unit to "Rel. Time", and save this setting by clicking "OK". Repeat for the 28S peak. For detailed information on how to define peak calling instructions, refer to Modifying peak calling instructions in the QIAxcel Advanced User Manual. QIAxcel RNA Handbook 06/

36 B4. Select samples and start peak calling. 36 QIAxcel RNA Handbook 06/2015

37 B5. Inspect the peak calling result in the peak calling result overview. Each row in this table represents the peak calling result for one sample. The RNA integrity score is given in the column "RIS". The 28S-to-18S-ratio is given in the column "Ratio". The total concentration of RNA is given in the column "Total RNA concentration". For the 18S and 28S peaks, it is listed whether or not a peak was found based on the criterion given in the peak calling instruction. For detailed information on the peak calling result table, refer to Peak Calling in the QIAxcel Advanced User Manual. Creating a RNA reference marker in QIAxcel ScreenGel Software To create a new reference marker table, a size maker analysis with annotated peak times and areas is necessary. A reference marker is formed by matching the peaks of the size marker data to a size marker table (including alignment marker). B1. In the Experiment Explorer, select the size marker channel. If the channel is not flagged as Size Marker in the software, right-click the size marker channel, and select the Size Marker option from the context menu that appears. B2. Open the size marker channel in the Single Electropherogram View. B3. Select an analysis profile in Analysis Properties in the Analysis tab or use the default parameters of the new analysis profile. If the profile is not visible, you can display it using the View menu (selecting the menu item View / Show Analysis Parameters ) or by clicking the icon at the extreme right of the view selection bar. B4. In Reference Marker, select No Marker. B5. Start the analysis by clicking the Start Analysis button. The result of the analysis is a sample with marked peaks. QIAxcel RNA Handbook 06/

38 B6. Check that the peaks were detected correctly. If necessary, correct with the Insert Peak and Delete Peak options. Note: If a size marker peak (usually the last) is not detected due to an increased baseline (leading to an increased threshold line), increase the Window parameter of the Baseline Filter until the baseline is nearly flat or decrease the Pos. threshold parameter. When an additional peak has been erroneously detected on a peak shoulder, delete incorrect peak using the Delete Peak option. B7. Open the Reference Marker tab, and select the alignment and size markers used. A comparison of the actual gel view (left) and the theoretical reference marker gel view (right) is displayed. When number of peaks in the actual size marker (left) matches the number of peaks in the reference marker (right), the reference marker can be used for sample analysis. 38 QIAxcel RNA Handbook 06/2015

39 B8. Optional (for ScreenGel Software version 1.0.x only): Define the tolerances for the detection of the alignment marker and the 18S and 28S fragments. The expected size of the fragments can be changed. Note: The expected sizes can be changed subsequently during sample analysis using the peaks of a sample. B9. Once the reference marker has been created, click the Apply button to copy it to Reference Marker in the Analysis tab. The reference marker can be saved for subsequent use by clicking the Save as... button. Note: The Apply and Save as... buttons are disabled and a warning is displayed at the top of the page when the reference marker is invalid (e.g., when the number of peaks for the reference and size markers does not match). If more than expected peaks were detected in the size marker, the extra peaks can be deleted using the Delete option. Alternatively, switch to the Single Electropherogram View and repeat the procedure from step 6. Data analysis with BioCalculator Software Before starting a RNA analysis, make sure the BioCalculator Software is in the RNA mode. When the RNA mode is active, the name of the software window is BioCalculator (RNA). To select the RNA mode, close all data files, select RNA from the Analysis menu (Figure 2), and open the data files for analysis. It is not possible to switch between DNA and RNA modes when data files are open. Figure 2. Switching to the RNA mode of BioCalculator Software. We recommend including the QX RNA Size Marker nt in at least 1 capillary when a gel cartridge is used for the first time. Size marker data can be used in subsequent runs; however, size and concentration calculations are more accurate when markers are included in each run. After the run, the Folder View (see Figure 3) displays the gel image as well as data for all channels and RNA-relevant analysis results. Data from an individual channel can be opened by double-clicking the channel in the gel image or by selecting the channel from the list at the right. QIAxcel RNA Handbook 06/

40 Figure 3. The Folder View. Procedure B1. Optional: Open the channel containing the RNA size marker by double-clicking the channel in the gel image or by selecting the channel from the list to the right. Note: If no RNA size marker was included in the run to be analyzed, continue with step B2. Size marker data from a previous run will be used. B2. Select Analysis from the BioCalculator menu bar and then select Reference Markers. The Reference Markers dialog box is displayed. B3. Load a size marker table from a file by clicking the Open button. Alternatively, click the Load Default button to load a size marker that has been set as the default in a previous run. The Set As Default and Load Default buttons enable reference marker table settings to be saved between runs without having to save them in a separate file. 40 QIAxcel RNA Handbook 06/2015

41 B4. Select the size marker channel from the drop-down list. The selected marker will be excluded from the results table. Note: If data from several runs (batches of 12) are analyzed in parallel, and the size marker is used in different positions in the runs, select N/A from the drop-down list. B5. Optional: Click the Fill button to load the size marker data (peak location and area) from the currently opened channel into the marker table. Note: If no RNA size marker was included in the run to be analyzed, continue with step B6. Size marker data from a previous run will be used. B6. Check Apply to all documents/folders, and click OK to start the analysis. B7. Switch to the folder view. B8. In the results table below the gel image, check whether the 18S and 28S alignment markers have been identified in each channel. If they have been identified, yes will appear in the corresponding cells. If not, the peaks can be identified manually and the analysis repeated (steps B9 11 below). B9. If all peaks are identified correctly, the analysis is complete. If one or more peaks are not identified correctly, select one of the channels containing total RNA. Right-click the alignment marker peak in the electropherogram, and select Marker from the drop-down list. B10. Right-click the 18S and 28S peaks to identify them manually. QIAxcel RNA Handbook 06/

42 B11. Open the reference marker table, and click OK to analyze the data again. To analyze data from a single channel, deselect the Apply to all documents/folders box. This step is necessary only after manual correction when channels with different parameters can not be analyzed together (for example, when RNA from different species is analyzed in the same run). If the migration times for 18S and 28S rrna vary considerably within a run, the Tol (%) values in the reference marker table can be adjusted to improve the identification; however, high tolerance % values may lead to misidentification of peaks. When finished, the data table should contain the estimated size for the ribosomal RNA fragments in nucleotides, an estimate of the total RNA concentration in ng/µl, and the 28S:18S rrna ratio. Note: Since migration times may vary slightly between different cartridges, we recommend saving a reference marker table for each new cartridge. Note: If more than 1 µl of RNA was added to the sample tube or if the RNA was diluted prior to measurement, correct the measurement accordingly when estimating RNA concentration. 42 QIAxcel RNA Handbook 06/2015

43 Appendix C: General Remarks on Handling RNA Handling RNA Ribonucleases (RNases) are very stable and active enzymes that generally do not require cofactors to function. Since RNases are difficult to inactivate and even minute amounts are sufficient to destroy RNA, do not use any plasticware or glassware without first eliminating possible RNase contamination. Great care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure. In order to create and maintain an RNase-free environment, the following precautions must be taken during pretreatment and use of disposable and nondisposable vessels and solutions while working with RNA. General handling Proper microbiological, aseptic technique should always be used when working with RNA. Hands and dust particles may carry bacteria and molds and are the most common sources of RNase contamination. Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin or from dusty laboratory equipment. Change gloves frequently and keep tubes closed whenever possible. Keep purified RNA on ice when aliquots are pipetted for downstream applications. To remove RNase contamination from bench surfaces, nondisposable plasticware, and laboratory equipment (e.g., pipets and electrophoresis tanks), use of RNaseKiller (cat. no ) from 5 PRIME ( is recommended. RNase contamination can alternatively be removed using general laboratory reagents. To decontaminate plasticware, rinse with 0.1 M NaOH, 1 mm EDTA* followed by RNase-free water (see Solutions, page 44), or rinse with chloroform* if the plasticware is chloroform-resistant. To decontaminate electrophoresis tanks, clean with detergent (e.g., 0.5% SDS),* rinse with RNase-free water, rinse with ethanol (if the tanks are ethanol resistant), and allow to dry. Disposable plasticware The use of sterile, disposable polypropylene tubes is recommended throughout the procedure. These tubes are generally RNase-free and do not require pretreatment to inactivate RNases. QIAxcel RNA Handbook 06/

44 Glassware Glassware should be treated before use to ensure that it is RNase-free. Glassware used for RNA work should be cleaned with a detergent,* thoroughly rinsed, and oven baked at 240 C for at least 4 hours (overnight, if more convenient) before use. Autoclaving alone will not fully inactivate many RNases. Alternatively, glassware can be treated with DEPC* (diethyl pyrocarbonate). Fill glassware with 0.1% DEPC (0.1% in water), allow to stand overnight (12 hours) at 37 C, and then autoclave or heat to 100 C for 15 minutes to eliminate residual DEPC (see Solutions, below). Solutions Solutions (water and other solutions)* should be treated with 0.1% DEPC. DEPC is a strong, but not absolute, inhibitor of RNases. It is commonly used at a concentration of 0.1% to inactivate RNases on glass or plasticware or to create RNase-free solutions and water. DEPC inactivates RNases by covalent modification. Add 0.1 ml DEPC to 100 ml of the solution to be treated and shake vigorously to bring the DEPC into solution. Let the solution incubate for 12 hours at 37 C. Autoclave for 15 minutes 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 2. When preparing Tris buffers, treat water with DEPC first, and then dissolve Tris to make the appropriate buffer. Trace amounts of DEPC will modify purine residues in RNA by carbethoxylation. Carbethoxylated RNA is translated with very low efficiency in cell-free systems. However, its ability to form DNA:RNA or RNA:RNA hybrids is not seriously affected unless a large fraction of the purine residues have been modified. Residual DEPC must always be eliminated from solutions or vessels by autoclaving or heating to 100 C for 15 minutes. * When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier. 44 QIAxcel RNA Handbook 06/2015

45 Appendix D: Removing Gel from Blocked Capillaries Blocked capillaries may lead to issues during calibration or sample runs. Gel can be removed from blocked capillaries by performing the standard geldroplet test. If the standard gel-droplet test fails, gel can be removed by performing the gel-droplet test with hot water or by performing the gel-droplet test using the QX Cartridge Prep Station. After confirmation of gel flow, a signal check can be performed before starting a calibration or sample run (see page 49). Standard gel-droplet test Note: The QIAxcel gel cartridge must be prepared before the standard geldroplet test is performed. Prepare the QIAxcel gel cartridge by following steps 1 4 on page 13. Procedure with QIAxcel ScreenGel Software D1. Launch the QIAxcel ScreenGel Software, and click Load Position in the status information bar. D2. Remove the buffer tray from the buffer tray holder. D3. Place a soft tissue on the buffer tray holder and click Park Position. D4. Install the QIAxcel gel cartridge and the smart key as described in Section of the QIAxcel User Manual or the QIAxcel Advanced User Manual. D5. Ensure that the cartridge identifier is displayed in the Cartridge Status window. D6. Click Latch to latch the cartridge if automatic latching is not active. D7. Switch to the Service Environment, and open the maintenance tab. D8. Click Long Purge to start purging. The purging process stops automatically after 3 min. To stop the purging process, click Stop. D9. Open the sample door. If all capillaries have formed homogeneous gel droplets, click Unlatch. Remove the gel cartridge from the instrument and carefully clean the capillary tips with a wet tissue. Note: If any capillary fails to form gel droplets, perform the gel-droplet test with hot water. D10. Remove the tissue from the buffer tray holder and insert the buffer tray into the buffer tray holder. D11. Place the gel cartridge into the QIAxcel instrument and perform the signal check (see page 49). QIAxcel RNA Handbook 06/

46 Procedure with BioCalculator Software D1. Launch the BioCalculator Software, and click Change Buffer in the Instrument Control window. D2. Remove the buffer tray from the buffer tray holder. D3. Place a soft tissue on the buffer tray holder, and click Park Position. D4. Install the QIAxcel gel cartridge and the smart key as described in Section of the QIAxcel User Manual. D5. Ensure that the cartridge identifier is displayed in the Instrument Control window. D6. Click Cart Latch. D7. Open File/Settings and then click Purge. The window shown below opens. 46 QIAxcel RNA Handbook 06/2015

47 D8. Leave the QIAxcel gel cartridge in the instrument (ignore the warning message), and click OK to start the purging process. The purging process stops automatically after 3 min. To stop the purging process, click Purge again. D9. Open the sample door. If all capillaries have formed homogeneous gel droplets, click Cart Unlatch. Remove the gel cartridge from the instrument and carefully clean the capillary tips with a wet tissue. Note: If any capillary fails to form gel droplets, perform the gel-droplet test with hot water. D10. Remove the tissue from the buffer tray holder and insert the buffer tray into the buffer tray holder. D11. Place the gel cartridge inside the QIAxcel instrument and perform the manual signal check (see page 49). Gel-droplet test with hot water If any of the capillaries fail to form droplets in the standard gel-droplet test, clogged capillaries can be cleared by performing a purge test with hot water. Procedure D1. If any of the capillaries fail to form droplets in the standard geldroplet test (page 45), fill the reservoir of the QX Cartridge Stand (cat. no ) with 12 ml hot (90 C or near boiling) water. Hot water QIAxcel RNA Handbook 06/

48 D2. Place the cartridge in the reservoir of the QX Cartridge Stand and submerge the tips for 7 10 min in the hot water. This should soften the dried gel at the capillary tips. D3. Empty the reservoir and fill again with 12 ml hot water. D4. Repeat step D2 to soften any dried gel at the capillary tips. D5. Place the cartridge into the QIAxcel instrument and perform the standard gel-droplet test again (see page 45). D6. Check whether gel droplets form at the capillary tips. If any of the capillaries still do not form homogeneous gel droplets, submerge the capillary tips in hot water for min and then repeat step D5. D7. If all capillaries form homogeneous gel droplets, clean the tips with wet tissue, and perform the manual signal check (see page 49). If after 3 attempts a capillary fails to form homogeneous gel droplets, contact QIAGEN Technical Services or perform the gel droplet test using the QX Cartridge Prep Station. Gel-droplet test using the QX Cartridge Prep Station If after 3 attempts homogeneous droplets do not form for all capillaries in the gel-droplet test with hot water (page 47), clogged capillaries can be cleared by performing a purge test in the QX Cartridge Prep Station (cat. no ). D1. Place the QIAxcel gel cartridge in the QX Cartridge Prep Station (cat. no ). D2. Attach the purge port clamp to the top of the cartridge (the knob should be in front of the cartridge), and gently tighten the knob to secure the cartridge. D3. Insert a QX Nitrogen Cylinder (cat. no ) into the pressure regulator (cat. no ), and secure it inside the cylinder stand. 48 QIAxcel RNA Handbook 06/2015

49 D4. Slowly adjust the pressure to psi, monitoring the pressure displayed in the pressure gauge. Do not exceed 65 psi. D5. Check to see whether gel droplets form at the capillary tips. D6. If all capillaries formed homogeneous gel droplets within 1 3 min, turn off the pressure, remove the purge clamp and clean the tips with wet tissue. D7. Place the cartridge into the instrument, and perform the manual signal check (see below). D8. If any of the capillaries fail to form gel droplets within 1 3 min, contact QIAGEN Technical Services. Performing a signal check The performance of the cartridge channels can be tested by performing a signal check. The signal check should be performed successfully (i.e., all channels should detect a single peak at minutes) before running the calibration wizard again. QIAxcel RNA Handbook 06/

50 Things to do before starting Prepare the buffer tray as described in Section of the QIAxcel User Manual or the QIAxcel Advanced User Manual. Load the buffer tray as described Section of the QIAxcel User Manual or the QIAxcel Advanced User Manual. Load 15 µl of the QX Intensity Calibration Marker into the MARKER2 position of the buffer tray (see Preparing the QIAxcel RNA gel cartridge and buffer tray, page 12 for more information). Procedure using QIAxcel ScreenGel Software D1. Launch the QIAxcel ScreenGel Software. D2. From the drop-down list in the process profile tab, select the Signal Check process profile that corresponds to the cartridge in use. D3. Click the Run Check tab, enter the required information, and click Run. D4. If a single peak is detected in all channels, intensity calibration can be performed again (see Intensity calibration, page 15). If one or more channels fail to detect a peak (i.e., no band present), contact QIAGEN Technical Services. Procedure using BioCalculator Software D1. Launch the BioCalculator Software. D2. Select the Cal.mtd method from the method drop-down list in the Instrument Control window. D3. To view the method parameters, select the Method tab. 50 QIAxcel RNA Handbook 06/2015

51 D4. To run the method, select the Sequence tab and click Run. D5. If a single peak is detected in all channels, perform intensity calibration (see Intensity calibration, page 15). If one or more channels fail to detect a peak (i.e., no band present), contact QIAGEN Technical Services. References QIAGEN maintains a large, up-to-date online database of scientific publications utilizing QIAGEN products. Comprehensive search options allow you to find the articles you need, either by a simple keyword search or by specifying the application, research area, title, etc. For a complete list of references, visit the QIAGEN Reference Database online at or contact QIAGEN Technical Services or your local distributor. QIAxcel RNA Handbook 06/

52 Ordering Information Product Contents Cat. no. QIAxcel RNA QC Kit v2.0 (1200) For 100 runs of 12 samples: QIAxcel RNA Quality Control Cartridge, Buffers, Mineral Oil, QX Intensity Calibration Marker, QX RNA Alignment Marker, QX RNA Size Marker nt, QX RNA Denaturation Buffer, 12-Tube Strips QIAxcel Advanced System Warranty PLUS 2 Basic, QIAxcel Warranty PLUS 2 Full, QIAxcel Software QIAxcel ScreenGel Software QIAxcel ScreenGel Software (10) QX Markers Capillary electrophoresis device, including computer, and QIAxcel ScreenGel Software; 1-year warranty on parts and labor 3-year warranty, response time of 5-working days, all labor, travel, and repair parts 3-year warranty, 48-hour (2 working days) priority response, all labor, travel, and repair parts Separate license for use of QIAxcel ScreenGel Software on an additional computer 10 separate licenses for use of QIAxcel ScreenGel Software on an additional computers QX RNA Size Marker nt (20 µl) QX RNA Alignment Marker (1.5 ml) QX Intensity Calibration Marker (600 µl) RNA size marker with 8 fragments: nt; concentration 60 ng/µl RNA alignment marker µl QX Intensity Calibration Marker QIAxcel RNA Handbook 06/2015

53 Product Contents Cat. no. QX Buffers QX RNA Denaturation Buffer (2 ml) QX RNA Dilution Buffer (15 ml) QX Separation Buffer (40 ml) QX Wash Buffer (40 ml) QX Mineral Oil (50 ml) 2ml QX RNA Denaturation Buffer ml QX RNA Dilution Buffer ml QX Separation Buffer ml QX Wash Buffer ml QX Mineral Oil Related products QIAxcel Kits for automated analysis of DNA or protein using the QIAxcel system QIAxcel DNA High Resolution Kit (1200) QIAxcel DNA Screening Kit (2400) QIAxcel DNA Fast Analysis Kit (3000) QIAxcel Protein Kit (1200) QIAxcel Accessories QX Cartridge Stand with Cover QIAxcel DNA High Resolution Cartridge, Buffers, Mineral Oil, QX Intensity Calibration Marker, 12-Tube Strips QIAxcel DNA Screening Cartridge, Buffers, Mineral Oil, QX Intensity Calibration Marker, 12-Tube Strips QIAxcel DNA Fast Analysis Cartridge, Buffers, Mineral Oil, QX Intensity Calibration Marker, QX DNA Size Marker 50 bp 1.5 kb, QX Alignment Marker 15 bp/3 kb, 12-Tube Strips QIAxcel Protein Gel Cartridge, Buffers, QX Mineral Oil, QX Pro Alignment Marker, QX Pro Marker kda, QX Pro Labeling Dye, QX Pro Calibration Marker, QX 0.2 ml 12-Tube Strips QX Cartridge Stand and QX Cartridge Stand Cover QIAxcel RNA Handbook 06/

54 Product Contents Cat. no. QX Cartridge Stand Cover QX Cartridge Stand Cover QX Buffer Tray Buffer tray for use with the QIAxcel system QX 0.2 ml 12-Tube Strip (80) QX Color 0.2 ml 12-Tube Strip (80) 80 x QX 0.2 ml 12-Tube Strips x QX Color 0.2 ml 12-Tube Strips QX 0.2 ml 12-Tube Strip Caps (80) QX Nitrogen Cylinder (6) 80 strip caps for use with QX 0.2 ml 12-Tube Strips QIAxcel Nitrogen Cylinders 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 or can be requested from QIAGEN Technical Services or your local distributor. 54 QIAxcel RNA Handbook 06/2015

55 Trademarks: QIAGEN, QIAxcel (QIAGEN Group). Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the QIAxcel RNA QC Kit v2.0 to the following terms: 1. The QIAxcel RNA QC Kit v2.0 may be used solely in accordance with the QIAxcel RNA Handbook and for use with components contained in the Kit only. QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the QIAxcel RNA Handbook and additional protocols available at 2. Other than expressly stated licenses, QIAGEN makes no warranty that this Kit and/or its use(s) do not infringe the rights of third-parties. 3. This Kit and its components are licensed for one-time use and may not be reused, refurbished, or resold. 4. QIAGEN specifically disclaims any other licenses, expressed or implied other than those expressly stated. 5. The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and/or its components. For updated license terms, see QIAGEN, all rights reserved.

56 Australia Austria Belgium Brazil Canada China Denmark Finland France Germany Hong Kong India Ireland Italy Japan Korea (South) Luxembourg Mexico The Netherlands Norway Singapore Sweden Switzerland UK USA /2015 Sample & Assay Technologies