QIAxcel RNA Handbook 3rd Edition April 2013

QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies, enabling the isolation and detection of contents of any biological sample. Our advanced, high-quality products and services ensure success from sample to result. QIAGEN sets standards in:



Purification of DNA, RNA, and proteins



Nucleic acid and protein assays



microRNA research and RNAi



Automation of sample and assay technologies

Our mission is to enable you to achieve outstanding success and breakthroughs. For more information, visit www.qiagen.com.

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 04/2013

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References

51

Ordering Information

52

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QIAxcel RNA Handbook 04/2013

Kit Contents QIAxcel RNA QC Kit v2.0

(1200)

Catalog no.

929104

Number of assays

12 x 100

QIAxcel RNA Quality Control Cartridge (with smart key)

1

QX Separation Buffer*

100 ml

QX Wash Buffer*

40 ml

QX Mineral Oil

50 ml

QX RNA Dilution Buffer

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 200–6000 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 200–6000 nt are delivered separately. QX RNA Size Marker 200–6000 nt should be stored at –20° to –80°C, and is stable for at least 6 months under these conditions.

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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 5.2.5 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 04/2013

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 www.qiagen.com).

Technical Assistance At QIAGEN, we pride ourselves on the quality and availability of our technical support. Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of QIAGEN products. If you have any questions or experience any difficulties regarding the 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 www.qiagen.com/Support or call one of the QIAGEN Technical Service Departments or local distributors (see back cover or visit www.qiagen.com).

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.

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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 www.qiagen.com/safety where you can find, view, and print the SDS for each QIAGEN kit and kit component. The following risk and safety phrases apply to components of the QIAxcel RNA QC Kit v2.0. QX RNA Denaturation Buffer Contains formaldehyde: irritant. Risk and safety phrases:* R43, S24-36/37/39-45 24-hour emergency information Chemical emergency or accident assistance is available 24 hours a day from: CHEMTREC USA & Canada  Tel: 1-800-424-9300 Outside USA & Canada  Tel: +1-703-527-3887 (collect calls accepted)

* R43: May cause sensitization by skin contact; S24: Avoid contact with the skin; S36/37/39: Wear suitable protective clothing, gloves, and eye/face protection; S45: In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).

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QIAxcel RNA Handbook 04/2013

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.

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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.)

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QIAxcel RNA Handbook 04/2013

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. 929703) 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 www.qiagen.com)



RNase-free water



PCR cycler, heat block, or water bath for denaturing RNA samples

For QIAxcel users



QIAxcel instrument (cat. no. 9001421) and BioCalculator Software or QIAxcel ScreenGel Software

For QIAxcel Advanced users



QIAxcel Advanced instrument (cat. no. 9001941) and QIAxcel ScreenGel Software

Note: If working with RNA for the first time, read Appendix C (page 43).

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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 15–20 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 6.5.1 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.

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QIAxcel RNA Handbook 04/2013

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.

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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.

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QIAxcel RNA Handbook 04/2013

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 SettingsAll UsersApplication DataQIAGENQIAxcelScreenGelDataCartridgeCalibrationData 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 04/2013

15

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 0.004–0.006. 4. If one or more channels show no signals in the first run, see Appendix D, page 45. 5. 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 FilesQIAxcel BioCalculator. A Calibration2.log file (cartridge calibration information) is saved automatically in the BioCalculator root directory: C:Program FilesQIAxcel 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.

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QIAxcel RNA Handbook 04/2013

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 0.004–0.006. 5. 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 45. 7. 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 200–6000 nt is used without dilution.

QIAxcel RNA Handbook 04/2013

17

Table 1. Suggested RNA concentrations Suggested concentration (ng/µl)

Method

300–1000

CM-RNA

50–300

CL-RNA

cRNA or singlestranded RNA

100–500

CM-RNA

<100

CL-RNA

Fragmented cRNA or cDNA

250–500

CM-F-RNA

RNA sample type Total RNA

2. Pipet RNA samples (1 µl total, ≤1 µg/µl) and QX RNA Size Marker 200–6000 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 6.3.3 of the QIAxcel Advanced User Manual.

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QIAxcel RNA Handbook 04/2013

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 6.3.3 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 12.

1. 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 5.2.3 of the QIAxcel User Manual or the QIAxcel Advanced User Manual for more details.

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5. Load the buffer tray containing the QX RNA Alignment Marker into the buffer tray holder. See Section 5.2.2 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 15–20 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.

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