Rotor-Gene Q, manual setup using Template Files in Q-Rex. Page 13. Rotor- Gene Q, setup with the QIAGEN Quantification Assay Data Handling and STR.
Investigator Quantiplex Kits
February 2018 Investigator® Quantiplex® Pro RGQ Kit Handbook For quantification of human and male DNA in forensic samples Sample to Insight__ Contents Kit Contents ............................................................................................................... 4 Storage ..................................................................................................................... 4 Intended Use .............................................................................................................. 4 Safety Information....................................................................................................... 5 Quality Control........................................................................................................... 5 Introduction................................................................................................................ 5 Principle and Procedure............................................................................................... 6 Internal control ................................................................................................. 7 Quantiplex Pro RGQ Reaction Mix ..................................................................... 8 Male Control DNA M1 and standard curve ......................................................... 9 Templates for routine work................................................................................. 9 Description of protocols..................................................................................... 9 Equipment and Reagents to Be Supplied by User .......................................................... 10 Important Notes........................................................................................................ 11 Selecting kits and protocols ............................................................................. 11 Contamination risks ........................................................................................ 11 Controls ........................................................................................................ 11 Protocol: Quantification of DNA Using Manual Setup and Template Files in Q-Rex and RotorGene Q................................................................................................................... 13 Protocol: Quantification of DNA Using the QIAGEN Quantification Assay Data Handling Tool and Rotor-Gene Q ............................................................................................. 24 Data analysis ........................................................................................................... 46 2 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Interpreting Data Using the QIAGEN Quantification Assay Data Handling Tool ............... 57 General Interpretation of Results ................................................................................. 63 General considerations for data analysis........................................................... 63 Standard curve............................................................................................... 63 Internal control ............................................................................................... 64 Quantification of unknowns ............................................................................. 65 Quantification of female/male mixtures............................................................. 65 Degradation status assessment ......................................................................... 66 Troubleshooting Guide .............................................................................................. 67 Appendix: Alternative Standard Curves ....................................................................... 70 Ordering Information ................................................................................................ 71 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 3 Kit Contents Investigator Quantiplex Pro RGQ Kit Catalog no. Number of 20 µl reactions Quantiplex Pro RGQ Reaction Mix Quantiplex Pro RGQ Primer Mix Male Control DNA M1 (50 ng/µl) QuantiTect® Nucleic Acid Dilution Buffer Storage (200) 387316 200 1 x 1.9 ml 1 x 1.9 ml 0.2 ml 1 vial Kit reagents should be stored immediately upon receipt at 30 to 15°C in a constanttemperature freezer. After first use, store the kit components at 28°C. Avoid freezing the kit components. The QuantiTect Nucleic Acid Dilution Buffer may also be stored at 30°C to 15°C, if desired. Quantiplex Pro RGQ Primer Mix must be stored protected from the light. DNA samples should be stored separately from PCR reagents. Under these conditions, the components are stable until the expiration date indicated on the kit. Intended Use The Investigator Quantiplex Pro RGQ Kit is intended for molecular biology applications in forensic, human identity and paternity testing. 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 that all users of QIAGEN® products adhere to the NIH guidelines that have been developed for recombinant DNA experiments, or to other applicable guidelines. 4 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 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. Quality Control In accordance with QIAGEN's ISO-certified Quality Management System, each lot of Investigator Quantiplex Pro RGQ kits is tested against predetermined specifications to ensure consistent product quality. Introduction Human identification is commonly based on the analysis of short tandem repeats (STRs), single nucleotide polymorphisms (SNPs) or deletion insertion polymorphisms (DIPs), depending on the demands of an examination or on the sample quality. These multiplex assays used for human identification are complex systems that require a defined range of template input. The Investigator Quantiplex Pro RGQ Kit provides quantification of human genomic DNA, male DNA and the integrity of DNA in a sample using quantitative real-time PCR. The kit is designed to confirm whether a sample contains sufficient DNA to enable DNA fingerprinting analysis (such as STR, DIP or SNP analysis). Furthermore, the kit may help in establishing whether a sample contains inhibitors that may interfere with such applications, thus Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 5 necessitating further sample purification. In addition, the newly developed DNA degradation systems allows for a more precise assessment of the degradation status of the DNA. The Investigator Quantiplex Pro RGQ Kit uses QuantiNova® DNA Polymerase, a novel hotstart enzyme, and QuantiNova Guard, a novel additive. These unique components further improve the stringency of the antibody-mediated hot-start. The kit also features a built-in control for visual identification for correct pipetting, and QBond®, an additive in the buffer that enables short cycling steps without loss of PCR sensitivity and efficiency. Principle and Procedure The Investigator Quantiplex Pro RGQ Kit is a ready-to-use system for the detection of human and male DNA, and parallel assessment of DNA degradation using quantitative real-time PCR. The kit provides fast and accurate quantification of human DNA in forensic database and casework samples. The kit contains reagents and a DNA polymerase for specific amplification of 4NS1C®, which is a 91 bp proprietary region present on several autosomes of the human genome, and for detection of the specific PCR products on the Rotor-Gene® Q System. The human quantification target region was selected to achieve high sensitivity. The human quantification target region is detected using the yellow channel on the Rotor-Gene Q. The target region for male DNA quantification was selected to achieve high sensitivity in the presence of mixed female/male DNA samples. The male quantification target region is detected as an 81 bp fragment using the green channel on the Rotor-Gene Q. In addition, the Investigator Quantiplex Pro RGQ Kit contains a balanced internal amplification control that is used to test successful amplification, and identify the presence of 6 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 PCR inhibitors. This heterologous amplification system is detected as a 434 bp internal control (IC), in the crimson channel, on the Rotor-Gene Q. Furthermore, the kit detects a longer autosomal amplification product (353 bp) targeting the same locus (4NS1C) as the 91 bp autosomal target. Due to the differently sized autosomal targets, the longer autosomal target is more susceptible to DNA degradation, allowing for a precise assessment of the degradation status of the DNA. The larger autosomal quantification target region is detected as a 353 bp fragment, using the red channel on the Rotor-Gene Q. A unique feature of the kit is that it detects a longer gonosomal amplification product (359 bp) targeting the same locus as the smaller 81 bp gonosomal male target. Due to the differently sized gonosomal targets, the longer gonosomal target is more susceptible to DNA degradation, allowing for a precise assessment of the degradation status of male DNA. The larger gonosomal quantification target region is detected as a 359 bp fragment, using the orange channel on the Rotor-Gene Q. Detection of amplification is performed using TaqMan® probes and a novel, fast PCR chemistry. Dual-labeled probes, such as TaqMan probes, contain a fluorescent reporter and a quencher at their 5' and 3' ends, respectively. During the extension phase of PCR, the 5' and 3' exonuclease activity of QuantiNova DNA Polymerase cleaves the fluorophore from the quencher. This results in detectable fluorescence that is proportional to the amount of accumulated PCR product. Internal control The Internal Control (IC) is amplified and detected in the crimson channel on the Rotor-Gene Q system. The IC is designed to be more sensitive to inhibitors than the human and male quantification targets. The comparison of the Cq values of the IC system for DNA standards with the Cq values of the IC system for unknown samples may provide an indication of potential inhibition of the reaction in the unknown samples. Therefore, even if the IC system Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 7 reports the presence of inhibitors in the sample, the DNA quantification will typically provide a reliable result. The presence of inhibitors in the sample may affect the downstream application and must be considered. Laboratory validation with relevant inhibitors should be performed to determine criteria for detecting inhibition. Quantiplex Pro RGQ Reaction Mix The Quantiplex Pro RGQ Reaction Mix contains the novel hot start QuantiNova DNA Polymerase and Quantiplex Pro RGQ reaction buffer. QuantiNova DNA Polymerase is provided in an inactive state and has no enzymatic activity at ambient or higher temperatures. The antibody-mediated hot-start mechanism prevents the formation and extension of nonspecific PCR products and primer-dimers during reaction setup and the first denaturation step. Therefore, this mechanism allows higher PCR specificity and accurate quantification. At low temperatures, the QuantiNova DNA Polymerase is kept in an inactive state by the QuantiNova Antibody and Guard, which stabilize the complex and improve the stringency of the hot start. After raising the temperature for 2 minutes to 95°C, the QuantiNova Antibody and Guard are denatured and the QuantiNova DNA Polymerase is activated, enabling PCR amplification. The hot start enables rapid and convenient roomtemperature setup. Furthermore, the specially developed Quantiplex Pro reaction buffer contains the additive QBond, which allows short cycling times on the Rotor-Gene Q. Q-Bond increases the affinity of the DNA polymerase for short, single-stranded DNA, reducing the time required for primer/probe annealing to a few seconds. In addition, the unique composition of the buffer supports the melting behavior of DNA, enabling short denaturation and annealing/extension times. The Quantiplex Pro RGQ Reaction Mix is also based on the unique QIAGEN PCR buffer system. The buffer contains a balanced combination of KCl and NH4Cl, which promotes a 8 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 high ratio of specific-to-nonspecific primer binding during the annealing step of each PCR cycle. This creates stringent primer annealing conditions, leading to increased PCR specificity. Male Control DNA M1 and standard curve DNA quantification standards are critical for accurate analysis. We strongly recommend a 27-fold dilution series with 4 concentration points in the standard curve, for each assay. The Control DNA contains pooled male DNA at a concentration of 50 ng/µl. To ensure pipetting accuracy, the minimum input volume of DNA for dilutions should be 5 µl. The standard curve is designed to be easily set up using a convenient 1:27 dilution series. If using QuantiTect Nucleic Acid Dilution Buffer to dilute the Control DNA, the dilutions are stable for at least 1 week at 28°C. Important: Male Control DNA M1 is optimized for use with the Investigator Quantiplex Pro RGQ kits only. Templates for routine work In order to streamline the instrument setup and the analysis of the results on the Rotor-Gene Q system, QIAGEN has developed a set of template files. Download the template files from the product resources page at www.qiagen.com/QPpro-rgq-template-files. Description of protocols Protocols for the Rotor-Gene Q system are provided in this handbook. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 9 Equipment and Reagents to Be Supplied by User When working with chemicals, always wear a suitable lab coat, disposable gloves and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier. Equipment Cooling device or ice Real-time thermal cycler Rotor-Gene Q system (5plex/6plex) with 72-Well Rotor/Rotor-Disc® 72 Rotor/RotorDisc 100 Rotor Q-Rex Software Q-Rex Absolute Quantification HID Plug-in Q-Rex QIAgility® Wizard Plug-in QIAGEN Quantification Assay Data Handling and STR Setup Tool Material Pipettes and pipette tips Strip Tubes and Caps, 0.1 ml (cat. no./ID: 981103 or cat. no./ID: 981106) Rotor-Disc 72; (cat. no./ID: 981301 or cat. no./ID: 981303) Rotor-Disc 100; (cat. no./ID: 981311 or cat. no./ID: 981313) Reagents Nuclease-free (RNase/DNase-free) consumables: special care should be taken to avoid nuclease contamination of all reagents and consumables used to set up PCR for sensitive detection of human DNA. 10 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Important Notes Selecting kits and protocols This handbook contains protocols and recommendations for DNA quantification using the Rotor-Gene Q (5plex/6plex), with protocols listed in Table 1. Real-time cyclers other than the Rotor-Gene Q (5plex/6plex) have not been validated by QIAGEN for DNA quantification, using the Investigator Quantiplex Pro RGQ Kit. Table 1. Protocols for the Investigator Quantiplex Pro RGQ Kit with Rotor-Gene Q Real-time thermal cycler Rotor-Gene Q, manual setup using Template Files in Q-Rex Rotor-Gene Q, setup with the QIAGEN Quantification Assay Data Handling and STR Setup Tool Protocol Page 13 Page 24 Contamination risks Do not remove the seal or caps of the reaction tubes once the amplification is complete. Removing the seal or caps increases the risk of contaminating subsequent reactions with amplified product. All reaction mixtures should be set up in an area separate from that used for DNA isolation and PCR product analysis (post-PCR), to minimize the potential for cross-contamination. Use disposable tips containing hydrophobic filters to minimize cross-contamination. Controls No-template control (NTC) Replicates of NTC reactions should be included in each quantification run to detect contamination. NTCs should contain all the components of the reaction, except for the Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 11 template. Quantification using the Investigator Quantiplex Pro RGQ Kit is highly sensitive; despite the fact that the reagents contained in the Quantiplex Kit undergo strict quality controls to assess that they are free of human DNA contamination, background DNA may be detected in rare cases due to the high sensitivity of the assay. Take great care to avoid contamination when pipetting the NTC. We recommend performing at least duplicate NTC reactions. Internal positive control An internal positive control (detected using a TaqMan probe) is used to test for successful amplification, and for the presence of PCR inhibitors. The primers, TaqMan probe and template for the internal control are all contained in the Quantiplex Pro RGQ Primer Mix. 12 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Protocol: Quantification of DNA Using Manual Setup and Template Files in Q-Rex and RotorGene Q This protocol is optimized for assay setup on the Rotor-Gene Q using Q-Rex Software 1.0 and template files for manual setup. For general instructions on instrument setup and Q-Rex software, refer to the Rotor-Gene Q User Manual and Q-Rex Software User Manual available on www.qiagen.com. Important points before starting Set up all reaction mixtures in an area separate from that used for DNA isolation and PCR product analysis (post-PCR). Use disposable tips containing hydrophobic filters to minimize cross-contamination. Use the cycling conditions specified in the protocol. The cycling is optimized for this assay. Use the template volume specified in the protocol. The reaction is optimized for use with 2 µl template DNA. Do not use more or less than 2 µl per 20 µl reaction. Dilutions of DNA quantification standards in QuantiTect Nucleic Acid Dilution Buffer can be stored between 2 and 8°C for at least 1 week. Optimal analysis settings are a prerequisite for accurate quantification data. Readjust the analysis settings (i.e., baseline settings and threshold values) for analysis of every reporter dye channel in every run. We recommend using the provided template files for manual setup in Q-Rex to streamline the instrument setup and the analysis of the results on the Rotor-Gene Q. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 13 Procedure: 1. Mix all solutions thoroughly before use to avoid localized concentrations of salt. 2. Prepare fresh serial dilutions of the Male Control DNA M1 according to Table 2. Vortex for at least 5 s, and centrifuge each dilution briefly before removing an aliquot for the next dilution. Use a new pipette tip for each dilution. Make sure not to introduce cross-contamination. Table 2. Serial dilutions of Male Control DNA M1 Serial dilution of Control DNA (ng/µl) 50 1.8519 0.0686 0.0025 Control DNA (µl) Undiluted DNA 5 5 5 QuantiTect Nucleic Acid Dilution Buffer (µl) 130 130 130 Note: Alternative standard curves are listed in "Appendix: Alternative Standard Curves", page 70. 3. Thaw the template nucleic acids. Mix all solutions thoroughly before use to avoid localized concentrations of salt. 4. Prepare a master mix according to Table 3. The master mix contains all of the components needed for PCR, besides the template (sample) DNA and nuclease-free water. Prepare a volume of master mix 10% greater than that required for the total number of PCR assays to be performed. This should include positive and negative control reactions. Reaction setup can usually be performed at room temperature (1525°C). However, we recommend keeping the reagents, samples and controls on ice or in a cooling device. 14 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Table 3. Master mix for DNA quantification Component Quantiplex Pro RGQ Reaction Mix Quantiplex Pro RGQ Primer Mix Total volume of master mix Volume per 20 µl reaction 9 µl 9 µl 18 µl Final concentration 1x 1x 5. Mix the master mix thoroughly, and dispense 18 µl into Rotor-Gene Q Tubes or into the Rotor-Disc. 6. Add 2 µl QuantiTect Nucleic Acid Dilution Buffer to the NTC tubes. Make sure that the NTC wells do not come into contact with human DNA. 7. Add 2 µl control DNA dilutions, or 2 µl unknown sample DNA, to the individual tubes and mix thoroughly. Close the tubes. Mix carefully to avoid localized concentrations of salt and DNA. Table 4 shows a possible tube setup. Make sure that the master mix and template are thoroughly mixed. It is required to run duplicates of the control DNA dilutions for each assay and on each reaction plate. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 15 Table 4. Possible setup of reactions on the Rotor-Gene Q using Strip Tubes Tube contents 1 50 9 NTC 17 UNK 25 UNK 33 UNK 41 UNK 49 UNK 57 UNK 65 UNK 2 50 10 NTC 18 UNK 26 UNK 34 UNK 42 UNK 50 UNK 58 UNK 66 UNK 3 1.8519 11 UNK 19 UNK 27 UNK 35 UNK 43 UNK 51 UNK 59 UNK 67 UNK 4 1.8519 12 UNK 20 UNK 28 UNK 36 UNK 44 UNK 52 UNK 60 UNK 68 UNK 5 0.0686 13 UNK 21 UNK 29 UNK 37 UNK 45 UNK 53 UNK 61 UNK 69 UNK 6 0.0686 14 UNK 22 UNK 30 UNK 38 UNK 46 UNK 54 UNK 62 UNK 70 UNK 7 0.0025 15 UNK 23 UNK 31 UNK 39 UNK 47 UNK 55 UNK 63 UNK 71 UNK 8 0.0025 16 UNK 24 UNK 32 UNK 40 UNK 48 UNK 56 UNK 64 UNK 72 UNK All amounts in ng/µl. UNK: Unknown sample. 8. Place closed PCR tubes in the appropriate rotor in the Rotor-Gene Q cycler and attach the locking ring. If you are using tubes, empty positions in the rotor should be filled with empty PCR tubes. 16 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 9. Open Q-Rex Software 1.0 and login as a user or as an administrator (for further details on how to do a first-time login on Q-Rex, about user roles or how to add new users, see Q-Rex Software User Manual available on www.qiagen.com). Figure 1. Setting up a quantification run in Q-Rex. 10.Select Create from other template (Figure 1). Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 17 Figure 2. Setting up a quantification run in Q-Rex. 11.Browse to locate the saved Quantiplex Pro RGQ template files for manual setup and select the correct Quantiplex Pro RGQ template file for 0.1 ml strip tubes, Rotor-Disc 72 or Rotor-Disc 100 (Figure 2). 12.Click on open (Figure 2). 18 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Figure 3. General settings in Q-Rex. 13.In the comments field add any additional information needed (e.g. kit lot number, kit expiry date, etc.) to be entered for the run (Figure 3). 14.Click on left side in the step marker on "Run Profile" (Figure 3). Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 19 Figure 4. Run profile settings in Q-Rex. 15.Confirm Rotor type and Reaction volumes" (Figure 4). 16.Confirm that the cycling conditions preset in the template file are the same as outlined in Table 5. 17.Click in the thermal profile in the area above the colored camera symbol (indicated by arrow in Figure 4). Acquisition settings will be displayed (Figure 5). 18.Click on the "Auto-gain" field for the green channel (Figure 5). 20 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Table 5. Cycling conditions for the Rotor-Gene Q Step Initial activation step Two-step cycling: Time 3 min Temperature 95°C Number of Comment cycles PCR requires an initial incubation at 95°C to activate the DNA polymerase. 40 Denaturation 5 s 95°C Combined annealing/extension 10 s 60°C Perform fluorescence data collection using the green, yellow, orange, red and crimson channels with auto-gain optimization, specified in table 6. Figure 5. Confirmation of auto-gain settings in Q-Rex. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 21 Figure 6. Confirm auto-gain settings on Q-rex. 19.Confirm that `'Auto-gain'' is selected (see Figure 6), and settings for the green channel preset in the template are the same as outlined in Table 6.'' Optimize gain'' should be selected before 1st acquisition. Confirm auto-gain settings also for the yellow, orange, red and crimson channels, according to Table 6. Table 6. Auto-gain settings for the different channels. Channel Green Yellow Orange Red Crimson Min. fluorescence 6 6 2 6 5 Max. fluorescence 8 8 4 8 7 Optimize gain Before 1st acquisition Before 1st acquisition Before 1st acquisition Before 1st acquisition Before 1st acquisition 22 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Figure 7. Entering sample information into Q-Rex. 20.Click on the left side in the step marker on "Sample Layout" (Figure 7). Confirm or edit sample layout for Standards and NTCs. 21.Enter samples IDs and set sample type. Sample type can be set/changed by right clicking on the "Type" field. Tubes that are not being used have to be marked as "Not in use". Select appropriate decimal format (Figure 7). 22.Start the Rotor-Gene cycler by clicking "Start run". 23.Proceed to data analysis on page 46. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 23 Protocol: Quantification of DNA Using the QIAGEN Quantification Assay Data Handling Tool and Rotor-Gene Q This protocol is optimized for assay setup with the QIAGEN Quantification Assay Data Handling Tool on the Rotor-Gene Q using Q-Rex Software 1.0 and template files for use with the Data Handling Tool. For general instructions on instrument setup and Q-Rex software, refer to the Rotor-Gene Q User Manual and Q-Rex Software User Manual available on www.qiagen.com. Important points before starting Set up all reaction mixtures in an area separate from that used for DNA isolation and PCR product analysis (post-PCR). Use disposable tips containing hydrophobic filters to minimize cross-contamination. Use the cycling conditions specified in the protocol. The cycling is optimized for this assay. Use the template volume specified in the protocol. The reaction is optimized for use with 2 µl template DNA. Do not use more or less than 2 µl per 20 µl reaction. Dilutions of DNA quantification standards in QuantiTect Nucleic Acid Dilution Buffer can be stored between 2 and 8°C for at least 1 week. Optimal analysis settings are a prerequisite for accurate quantification data. Readjust the analysis settings (i.e., baseline settings and threshold values) for analysis of every reporter dye channel in every run. Note: This protocol requires the Q-Rex QIAgility Wizard Plug-in to be installed. If the QRex QIAgility Plug-in is not installed proceed to protocol on page 13. 24 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Procedure: 1. Mix all solutions thoroughly before use to avoid localized concentration of salt. 2. Prepare fresh serial dilutions of the Male Control DNA M1 according to Table 7. Vortex for at least 5 s, and centrifuge each dilution briefly before removing an aliquot for the next dilution. Use a new pipette tip for each dilution. Make sure to not introduce cross-contamination. Table 7. Serial dilutions of Male Control DNA M1 Serial dilution of Control DNA (ng/µl) 50 1.8519 0.0686 0.0025 Control DNA (µl) Undiluted DNA 5 5 5 QuantiTect Nucleic Acid Dilution Buffer (µl) 130 130 130 Note: Alternative standard curves are listed in "Appendix: Alternative Standard Curves", page 70. 3. Thaw the template nucleic acids. Mix all solutions thoroughly before use to avoid localized concentration of salt. 4. Prepare a master mix according to Table 8. The master mix contains all of the components needed for PCR besides the template (sample) DNA and nuclease-free water. Prepare a volume of master mix 10% greater than that required for the total number of PCR assays to be performed. This should include positive and negative control reactions. Reaction setup can usually be done at room temperature (1525°C). However, we recommend keeping the reagents, samples and controls on ice, or in a cooling device. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 25 Table 8. Master mix for DNA quantification Component Quantiplex Pro RGQ Reaction Mix Quantiplex Pro RGQ Primer Mix Total volume of master mix Volume per 20 µl reaction 9 µl 9 µl 18 µl Final concentration 1x 1x 5. Mix the master mix thoroughly, and dispense 18 µl into Rotor-Gene Q Tubes or into the Rotor-Disc. 6. Add 2 µl QuantiTect Nucleic Acid Dilution Buffer to the NTC tubes. Make sure that the NTC tubes do not come in contact with human DNA. 7. Add 2 µl control DNA dilutions, or 2 µl unknown sample DNA, to the individual tubes and mix thoroughly. Close the tubes. Mix carefully in order to avoid localized concentration of salt. Table 9 shows a possible tube setup. Make sure that the master mix and template are thoroughly mixed. It is required to run duplicates of the control DNA dilutions for each assay and for each run. 26 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Table 9. Possible setup of reactions on the Rotor-Gene Q using Strip Tubes. Tube contents 1 50 9 NTC 17 UNK 25 UNK 33 UNK 41 UNK 49 UNK 57 UNK 65 UNK 2 50 10 NTC 18 UNK 26 UNK 34 UNK 42 UNK 50 UNK 58 UNK 66 UNK 3 1.8519 11 UNK 19 UNK 27 UNK 35 UNK 43 UNK 51 UNK 59 UNK 67 UNK 4 1.8519 12 UNK 20 UNK 28 UNK 36 UNK 44 UNK 52 UNK 60 UNK 68 UNK 5 0.0686 13 UNK 21 UNK 29 UNK 37 UNK 45 UNK 53 UNK 61 UNK 69 UNK 6 0.0686 14 UNK 22 UNK 30 UNK 38 UNK 46 UNK 54 UNK 62 UNK 70 UNK 7 0.0025 15 UNK 23 UNK 31 UNK 39 UNK 47 UNK 55 UNK 63 UNK 71 UNK 8 0.0025 16 UNK 24 UNK 32 UNK 40 UNK 48 UNK 56 UNK 64 UNK 72 UNK All amounts in ng/µl. UNK: Unknown sample. 8. Place closed PCR tubes in the appropriate rotor in the Rotor-Gene Q cycler, and attach the locking ring. If you are using tubes, empty positions in the rotor should be filled with empty PCR tubes. 9. Open the QIAGEN Quantification Assay Data Handling and STR Setup Tool. 10.Click the `'Configuration'' worksheet tab (Figure 8). Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 27 Figure 8. Opening the `'Configuration'' worksheet. 11.Set the root/home directory to save Quant batch files (Figure 9). 12.Set the root/home directory to import Quant result files (Figure 9). 13.Set cycler dependent default target naming and tube setup for RGQ (Figure 10). 28 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Figure 9. Setting the Quant batch, Quant result files, tube setup and analysis criteria. Figure 10. Setting cycler dependent default target naming and tube setup for RGQ. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 29 14.Click the `'Quantification Setup'' worksheet tab (Figure 11). Figure 11. Setting the `'Quantification Setup''. 30 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Figure 12. Setting the `'Quantification Setup''. 15.Select RGQ instrument in the drop down menu (Figure 12). 16.Select Assay Quantiplex Pro RGQ (Figure 12). 17.Enter your Run ID for the run (Figure 12). 18.Select plasticware: Strip Tubes and Caps Rotor-Disc 72 Rotor-Disc 100 19.Optionally enter kit lot number and kit expiry date (Figure 12). Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 31 Figure 13. Inserting standards and sample IDs. 20. Click in the sample ID field (e. g. tube 1) to enter the DNA standards (Figure 13). 21.Then, click on the button "Insert Standards" and choose the layout for the standards (Figure 13). 22.Enter further sample IDs for each tube used (Figure 14). 23.Finish `'Quantification Setup'' by clicking "Create Batch file" (Figure 15). The Quant Batch file will be created in the root directory specified in step 11 (Figure 9). Use this Quant Batch file for simple run setup in Q-Rex. Proceed to step 24. 32 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Figure 14. Inserting standards and sample IDs. Figure 15. Creating a Quant Batch file. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 33 24.Open Q-Rex Software 1.0 and login as a user or as an administrator (for further details on how to do a first time login in Q-Rex, about user roles or how to add new users, see Q-Rex Software User Manual available on www.qiagen.com). Figure 16. Setting up a quantification run in Q-Rex. 25.Select QIAgility Wizard (Figure 16). Note: To import the Quant Batch file into Q-Rex the Q-Rex QIAgility Wizard Plug-in 1.0.1 needs to be installed. The plugin is available on www.qiagen.com. 34 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Figure 17. Selecting the Quant Batch file. 26.Select the Quant Batch file created in step 23 (Figure 17). Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 35 Figure 18. Selecting the Quant Batch file. 27.Click on next (Figure 18) and browse to locate the saved Quantiplex Pro RGQ Template files for use with the Data Handling Tool and select the correct Quantiplex Pro RGQ Template file for 0.1 ml strip tubes, Rotor-Disc 72 or Rotor-Disc 100 (Figure 19). 28.Click on open (Figure 19). 36 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Figure 19. Selecting the Quantiplex Pro RGQ Template Q-Rex. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 37 Figure 20. Selecting the Quantiplex Pro RGQ Template Q-Rex. 29.Confirm kit information, Q-Rex Template and click next (Figure 20). 38 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Figure 21. Assigning target names and acquisition channels. 30.Target names are automatically assigned to acquisition channels; click on finish (Figure 21). Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 39 Figure 22. General settings in Q-Rex. 31.In the comments field add any additional information needed to be entered for the run (Figure 22). 32.Click on left side in the step marker on "Run Profile" (Figure 22). 40 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Figure 23. Run profile settings in Q-Rex. 33.Confirm Rotor type and Reaction volumes" (Figure 23). 34.Confirm that the cycling conditions preset in the template file are the same as outlined in Table 10. 35.Click in the thermal profile in the area above the colored camera symbol (indicated by arrow in Figure 23). Acquisition settings will be displayed (Figure 24). 36.Click on the "Auto-gain" field for the green channel (Figure 24). Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 41 Table 10. Cycling conditions for the Rotor-Gene Q Step Initial activation step Two-step cycling: Time 3 min Temperature 95°C Number of cycles 40 Comment PCR requires an initial incubation at 95°C to activate the DNA polymerase. Denaturation 5 s 95°C Combined annealing/extension 10 s 60°C Perform fluorescence data collection using the green, yellow, orange, red and crimson channels with auto-gain optimization specified in table 11. Figure 24. Confirmation of Auto-gain settings in Q-Rex. 42 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Figure 25. Confirm auto-gain settings in Q-Rex. 37.Confirm that `'Auto-gain'' is selected, and settings for the green channel preset in the template are the same as outlined in Table 11. Optimize gain should be selected before 1st acquisition. Also confirm auto-gain settings for the yellow, orange, red and crimson channels according to Table 11. Table 11. Auto-gain Settings for the different channels. Channel Green Yellow Orange Red Crimson Min fluorescence 6 6 2 6 5 Max fluorescence 8 8 4 8 7 Optimize gain Before 1st acquisition Before 1st acquisition Before 1st acquisition Before 1st acquisition Before 1st acquisition Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 43 Figure 26. Confirmation of sample settings in Q-Rex. 38.Click on left side in the step marker on "Sample Layout" (Figure 26). Confirm sample layout (Standards, NTCs and Samples). Tubes that are not being used should be marked as "Not in use". Sample type can be changed by right clicking on the "Type" field. Select appropriate decimal format (Figure 26). Note: For tubes marked as "Not in use" a standard acquisition target (green, yellow, orange, red and crimson) can be added in addition to Male, Human, Human Degradation, Male Degradation and IC. Please check if additional targets have been added and remove the additional targets for "Not in use" tubes, as described in step 39, otherwise proceed to step 40. 44 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 39.Scroll down in the `'Define targets" windows and remove the additional targets (green, yellow, orange, red and crimson) assigned for `'Not in use" tubes by clicking on the red delete field (Figure 27). Take care not to remove the imported targets Male, Human, Male Degradation, Human Degradation and IC. Figure 27. Remove additional targets for "Not in use" tubes. 40.Start the Rotor-Gene cycler by clicking "Start run". 41.Proceed to data analysis on page 46. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 45 Data analysis Optimal analysis settings are a prerequisite for accurate quantification data. Readjust the analysis settings (i.e., baseline settings and threshold values) for analysis of every reporter dye channel in every run. Procedure 1. Open the run file using the Q-Rex software. Go to "File", followed by "Open Experiment and then browse to locate the saved run file. 2. Standards must be defined before a standard curve can be created. If the standards were defined before the run was started, proceed to step 3, otherwise edit sample layout as described on page 44, Figure 26. 46 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Figure 28. Setting analysis parameters in Q-Rex. 3. Click on the left side in the step marker on "Analysis" (Figure 28). Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 47 Figure 29. Setting analysis method in Q-Rex. 4. Confirm that the correct analysis method is selected (Absolute Quantification HID; Figure 29). If yes proceed to step 6, if not proceed to next step. 5. Add analysis method by clicking on the selection menu within the "Analysis" step marker on the left side, followed by "Add analysis" and then selecting `'Absolute Quantification HID'' (Figure 30). 48 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Figure 30. Selecting `'Absolute Quantification HID'' in Q-Rex. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 49 Figure 31. Setting analysis parameters in Q-Rex. 6. Select the green channel (Male target) within the normalized data section (Figure 31). 7. Select "Filter data" on the right panel (Figure 31). 8. Confirm selection of "Remove non-amplified curves with" and enter following values: for "fluorescence change" <7% for "reaction efficiency" < -100% 9. Select "Normalization'' (Figure 31). 10.Confirm that "Dynamic tube", "Use noise slope correction", "Ignore first cycles = set to 5" are selected and set. 50 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 11.Confirm "Adjust take-off points" is enabled and set to: if take-off point <12 use take-off cycle 20. 12.Select "Cq calculation" on the right panel (Figure 31). 13.Confirm `'Threshold'' is set to 0.008 and `'Threshold start cycle'' is set to 5. 14.Repeat step 7 to 14 for the yellow (Human target), orange (Male Degradation target), red (Human Degradation target) and crimson (IC) channel, and confirm analysis settings according to table 12. Table 12. Analysis settings for the different channels on Rotor-Gene Q Parameters Filter data "Remove non-amplified curves'' fluorescence change reaction efficiency Normalization Dynamic tube Use noise slope correction Ignore first cycles Adjust take-off points If take-off point Use take-off cycle Cq calculation Threshold Threshold start cycle Green Yellow Orange Red Crimson <7% <4% <2% <5% NA < -100% < -100% < -100% < -100% NA Enable Enable 5 Enable 12 20 Enable Enable 5 Enable 12 20 Enable Enable 5 Enable 12 20 Enable Enable 5 Enable 12 20 Enable Enable* 2 Enable 10 20 0.008 5 0.02 5 0.03 5 0.02 5 0.03 5 *For older Corbett Rotor Gene 6000 5plex Instruments disable "Use noise slope correction" for the IC in the crimson channel. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 51 Figure 32. Standard curves in Q-Rex. 15.The Standard Curves are shown in the "Targets" windows. Select one or multiple targets. View the calculated regression line, slope (M), y-intercept (B), R2 and efficiency values by clicking on the fx-symbol (Figure 32). 52 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Figure 33. Tube selector in Q-Rex. 16.Individual tubes can be selected or deselected for further analysis by using the "Tube selector" (Figure 33). View the concentration of the unknown samples in the tube table section on the left panel (Figure 33). All windows can be enlarged by clicking on the enlarge button (Figure 33). 17.To export the results in .csv format for further analysis with the QIAGEN Quantification Assay Data Handling Tool, click on the left side in the step marker on "Report/Export" (Figure 34). Note: A standard curve needs to be set and defined in order to export calculated concentrations for unknown samples. Only selected tubes will be exported. All channels/targets need to have a valid threshold set. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 53 Figure 34. Exporting data in Q-Rex. 18.Confirm that the analysis method "Absolute Quantification HID" has been selected (Figure 34). 19.Select `'Export sheet'' (Figure 35). 54 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Figure 35. Exporting data to Q-Rex. 20.Confirm export format as "Absolute Quantification HID Analysis Export'' (Figure 35). 21.Click `'Export'' (Figure 35). Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 55 Figure 36. Selecting export directory in Q-Rex. 22.Choose directory for data export and click ok (Figure 36). 23.To interpret the results, see "Interpreting Data Using the QIAGEN Quantification Assay Data Handling Tool" on page 57. 56 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Interpreting Data Using the QIAGEN Quantification Assay Data Handling Tool The QIAGEN Quantification Assay Data Handling Tool is designed for accurate quantification, data analysis and interpretation. The Opening Page worksheet contains information on version number and software requirements/compatibility. On the Configuration worksheet, the root directories for data processing, result import options and default values for analysis criteria and thresholds can be set. Each worksheet contains an instruction button, which, when pressed, provides detailed instructions on using the functions of the specific worksheet. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 57 Procedure: 1. Open the QIAGEN Quantification Assay Data Handling Tool. Click the `'Configuration'' worksheet tab (Figure 37). Figure 37. Opening the `'Configuration'' worksheet. 2. Set the root directory to save Quant batch files (Figure 38). 3. Set the root directory to import Quant result files (Figure 38). 58 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Figure 38. Setting directories for the Quant batch and Quant results files. 4. Target names should be assigned for the Rotor-Gene Q. Default names for the targets are "Human" (Human Target), "Male" (Male Target), "Human Degradation "(Human Degradation Target), Male Degradation (Male Degradation Target) and "IC" (Internal Positive Control). Defaults can be restored for Rotor-Gene Q by clicking the "Enter defaults" button (Figure 38). 5. Threshold setting for the Quality Assessment can be changed/adjusted as needed. The default threshold settings are Mixture index (Human/Male): 2 Degradation Index (Human/Degradation): 10 Inhibition Index (IC Shift): 1 Male Degradation Index (Male/Male Degradation): 10 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 59 Note: Setting the appropriate threshold values may require further internal validation at your facility. 9948 will be filtered from the import providing it is included in the section "Quantification QC Control Specification". Removing it allows it to be kept in the final data set. Defaults can be restored by clicking the `'Enter defaults'' button. 6. To import quantification results click the `'Quant Result Import'' worksheet tab (Figure 39). Figure 39. Opening the `'Quant Result'' Import tab. 7. To import your quantification results, click the `'Import QuantData'' button (Figure 40). 60 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Figure 40. Quant Result Import worksheet: Importing quantification results. 8. Confirm that your data are in the necessary format (Figure 41). Figure 41. `'Quant Result Import'' worksheet: Confirming your data format. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 61 9. Your quantification data are now imported and the data is analyzed. The Mixture Index, Degradation Indices and Inhibition Index are calculated and tagged as "Below Threshold", "Possible Mixture", "Possible Degradation" or "Possible Inhibition" (Figure 42). Figure 42. `'Quant Result Import'' worksheet: data analyzed. 10.Display options can be adjusted by clicking on `'Display settings'': Show Raw Data Show Quantity Mean Values Show Cq Values Note: The Degradation Indices for Human and Male are set to 10, as a default. Full STR profiles can be obtained with DNA fragmented to an average fragment size of approximately 300 bp. The default Human Degradation and Male Degradation Indices of 10 should allow differentiation between DNA fragments larger or smaller than 300 bp. 62 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Note: The default Inhibition Index is set to 1, as a default. The IC acts as a quality sensor and reports the presence of inhibitors with a Cq shift while quantification remains reliable. The default value can be changed and adjusted for relevant degrees of inhibition. Hence laboratory validation should be performed to determine criteria for detecting inhibition. General Interpretation of Results General considerations for data analysis Real-time PCR data are produced as sigmoidal-shaped amplification plots (when using a linear scale), in which fluorescence is plotted against the number of cycles. The threshold cycle (Cq value) serves as a tool for calculation of the starting template amount in each sample. This is the cycle in which there is the first detectable significant increase in fluorescence. The optimal threshold setting depends on the reaction chemistries used for PCR. Therefore, an optimal threshold setting that has been established for another kit may not be suitable for the Investigator Quantiplex Pro RGQ Kit and may need to be adjusted. For DNA quantification using the Investigator Quantiplex Pro RGQ Kit, the analysis settings must be adjusted for both reporter dyes. Standard curve The standard curve is the best fit for a linear regression to the standard dilution series data. The equation is in the form y = mx + b Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 63 where x = log concentration and y = Cq. The slope The slope (m) describes the PCR efficiency. A slope of 3.3 indicates 100% PCR efficiency (i.e., the number of copies of amplification product is doubled in each cycle). Typically, the slope ranges between -3.0 and -3.6. If the values fall outside of this range, see the Troubleshooting Guide, page 67, for more information. The Y-intercept The Y-intercept (b) indicates the expected Cq value for a sample with a quantity value of 1 (for example, 1 ng/µl). The R2 value The R2 value is a measure of the fit of the data points to the regressed line. In general, the standard curve has an R2 value of 0.990. Low R2 values (R2 0.98) may occur for many different reasons. In case of low R2 values, see the Troubleshooting Guide, page 67, for more information. Internal control The internal control (IC)is intended to report chemistry or instrument failure, errors in assay setup and the presence of inhibition in the sample. The IC system is designed to be more sensitive to inhibition than the specific target for human DNA. Therefore, the quantification will be valid even if some inhibition is present in the sample. In this case, the operator will get information both about the concentration of DNA in the sample and about the presence of inhibitors. Comparison of the Cq value of the IC system for DNA standards with the Cq values of the IC system for unknown samples can provide an indication of potential inhibition. At higher concentrations of inhibitor, the quantification data may be affected, and this must be considered for downstream applications. In general, the internal control can be interpreted in the following manner. 64 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 a) IC system shows normal amplification No IC shift greater than specified is observed No amplification of the Human, Degradation and Male Targets is detectable No or insufficient DNA was present. b) IC shift is greater than specified Degradation Index is below threshold Sample contains inhibitors. DNA is not degraded. c) IC shift is greater than specified Degradation Index is above threshold Sample contains inhibitors. DNA is possibly degraded. Note: Extremely high concentrations of inhibitors can inhibit amplification of the degradation target and trigger the Degradation Index. Important: Internal laboratory validation with relevant inhibitors should be performed to determine criteria for detecting inhibition. Quantification of unknowns The Investigator Quantiplex Pro RGQ Kit can quantify a broad range of DNA amounts in a sample, from 200 ng/µl to approximately 0.5 pg/µl of human genomic DNA. When 2 µl of a sample with very low concentrations is loaded in a reaction, the well probably contains less than one diploid human genome equivalent. In the low DNA concentration range, statistical effects, known as stochastic variations, can significantly affect the assay result. When using samples with low concentrations of DNA, make sure that as many replicates as possible are assayed in order to confirm the result. Quantification of female/male mixtures The Investigator Quantiplex Pro RGQ Kit provides high sensitivity to detect low amounts of male DNA, even in a very high background of female DNA. The Mixture Index provides information on whether a sample is a female/male mixture. In general, the Mixture Index can be interpreted in the following manner. a) The sample has a Mixture Threshold below the index specified b) The sample has a Mixture Threshold above the index specified The sample contains only male DNA or only low levels of female DNA. The sample contains a possible male DNA/female DNA mixture. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 65 Degradation status assessment Environmental degradation may occur with forensic casework samples and is a typical challenge in routine genetic fingerprinting. The Investigator Quantiplex Pro RGQ Kit contains a newly developed system for detection of human DNA degradation in general and male specific DNA degradation in mixture samples. In general, the Degradation Indices can be interpreted in the following manner. a) The sample has a Degradation Threshold below the index specified No IC shift is detected b) The sample has a Degradation Threshold below the index specified IC shift is detectable above the threshold c) The sample has a Degradation Threshold above the index specified No IC shift is detected d) The sample has a Degradation Threshold above the index specified IC shift is detectable above the threshold DNA is most likely not degraded. The sample most likely contains no inhibitors. DNA is most likely not degraded. The sample may contain inhibitors. DNA is most likely degraded. The sample most likely contains no inhibitors. DNA may or may not be degraded. The sample contains inhibitors. Note: When 2 µl of a sample with very low concentrations is loaded in a reaction, the well probably contains less than one diploid human genome equivalent. In the low DNA concentration range, statistical effects, known as stochastic variations, can affect the assay. In case of degraded DNA with a very low DNA concentration, the Degradation targets can be affected. If the Degradation targets have an undetermined value, the sample will be tagged with "Possible Degradation". Extremely high inhibitor concentrations can also affect the Degradation targets and lead to a "Possible Degradation" flag. 66 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise. For more information, see also the Frequently Asked Questions page at our Technical Support Center: www.qiagen.com/FAQ/FAQList.aspx. The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies (for contact information, see back cover or visit www.qiagen.com). Comments and suggestions No signal or one or more signals detected late in PCR a) Incorrect cycling conditions Always use the optimized cycling conditions specified in the protocols. Make sure to select ROX as the passive dye on Applied Biosystems® instruments. b) Pipetting error, missing or degraded reagent Check the storage conditions of the reagents. Repeat the assay. c) Incorrect or no detection step Make sure that fluorescence detection takes place during the combined annealing/extension step. d) Insufficient amount of starting template Increase the amount of template, if possible. Ensure that sufficient copies of the template DNA are present in the sample. e) Problems with starting template f) Wrong detection channel/filter chosen g) Degraded control DNA Check the storage conditions of the starting template DNA. Efficient removal of PCR inhibitors is essential for optimal results. Purify nucleic acids from your sample using an appropriate purification method. Ensure that all reagents, buffers and solutions used for isolating and diluting template nucleic acids are free from nucleases. Ensure that the correct detection channel is activated or the correct filter set is chosen for each reporter dye. Ensure that the chosen combination of reporter dyes is compatible with the selected detection channels or filter sets. Make new serial dilutions of the control DNA from the stock solution. Repeat the assay using the new dilutions. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 67 Comments and suggestions Differences in Cq values or in PCR efficiencies between runs a) Incorrect cycling conditions Always start with the optimized cycling conditions specified in the protocols. Ensure that the cycling conditions include the initial step for activation of the DNA polymerase and the specified times for denaturation and annealing/extension. b) Analysis settings (e.g., threshold and baseline settings) not optimal Check the analysis settings (threshold and baseline settings) for each reporter dye. Repeat analysis using optimal settings for each reporter dye. No linearity in ratio of Cq value/crossing point to log of the template amount Amount of template in unknown sample too high Linearity is guaranteed within the range of the standard curve. If signals appear at very early Cq values, dilute the sample and repeat the reaction. Increased fluorescence or Cq value for no-template control a) Contamination of reagents Discard all the components of the assay (e.g., master mix). Repeat the assay using new components. b) Minimal probe degradation, leading to sliding increase in fluorescence Check the amplification plots, and adjust the threshold settings. c) Crosstalk problems Varying fluorescence intensity a) Contamination of real- time cycler b) Real-time cycler no longer calibrated Depending on the instrument, different techniques are used to avoid spectral crosstalk when using multiple fluorophores for multiplex assays. However, minimal crosstalk, as a result of residual spectral overlap, may be observed in the NTC wells, especially if the instrument is in need of calibration. Reactions were contaminated with target DNA. Decontaminate the real-time workstations and the cycler according to the manufacturer's instructions. Use new reagents and solutions. Recalibrate the real-time cycler according to the manufacturer's instructions. c) Wavy curve at high template amounts for highly concentrated targets In the analysis settings, reduce the number of cycles used for background calculation (if the real-time cycler allows this) or reduce the amount of template. 68 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Comments and suggestions Slope for the standard curve differs significantly from 3.33 or R2 value is significantly less than 0.980.99 a) Contamination of realtime cycler Decontaminate the real-time cycler according to the manufacturer's instructions. b) Real-time cycler and/or pipettes no longer calibrated Recalibrate the real-time cycler according to the manufacturer's instructions. Calibrate pipettes to minimize pipetting variability. c) Wavy curve at high template amounts for highly concentrated targets In the analysis settings, reduce the number of cycles used for background calculation or reduce the amount of template. d) Problem with dilution of standards e) Plate not sealed Ensure that the DNA standard is completely thawed and mixed thoroughly before use. Ensure that dilutions of the DNA standard are mixed thoroughly before removing each aliquot for the serial dilution. Always use a sample volume of 2 µl. Change pipette tips between each dilution step. Carefully seal the plates to avoid evaporation. f) Error made during dilution of the DNA standard Verify all calculations, and repeat dilution of the DNA standard. g) Incorrect concentration values entered in the software Verify the concentrations for all samples used to generate the standard curve. h) Abnormal fluorescence Do not write on the plate. Use caution when handling plates. Wear gloves. i) Statistical variation Some variation in the reaction is normal, particularly when the DNA target is present at a low copy number. Perform at least duplicates for the standard curve to minimize the effect of this variation. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 69 Appendix: Alternative Standard Curves Table 13. Alternative 5-point standard curve (10x dilution) Serial dilution of control DNA (ng/µl) 50 5 0.5 0.05 0.005 Amount of control DNA (µl) QuantiTect Nucleic Acid Dilution Buffer (µl) Undiluted DNA 5 45 5 45 5 45 5 45 Table 14. Alternative 6-point standard curve (9x dilution) Serial dilution of control DNA (ng/µl) 50 7.1429 1.0204 0.0686 0.0076 0.0030 Amount of control DNA (µl) QuantiTect Nucleic Acid Dilution Buffer (µl) Undiluted DNA 5 40 5 40 5 40 5 40 5 40 Table 15. Alternative 7-point standard curve (5x dilution) Serial dilution of control DNA (ng/µl) 50 10 2 0.4 0.08 0.016 0.0032 Amount of control DNA (µl) QuantiTect Nucleic Acid Dilution Buffer (µl) Undiluted DNA 10 40 10 40 10 40 10 40 10 40 10 40 70 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Ordering Information Product Contents Investigator Quantiplex Pro RGQ Kit (200) Reaction Mix, Primer Mix, Control DNA, QuantiTect Nucleic Acid Dilution Buffer Cat. no. 387316 Related products Investigator human identification PCR kits Investigator Quantiplex Kit (200) Reaction Mix FQ, Primer Mix IC FQ, Control DNA Z1, QuantiTect Nucleic Acid Dilution Buffer Investigator Quantiplex HYres Kit (200) Reaction Mix YQ, Primer Mix IC YQ, Control DNA Z1, QuantiTect Nucleic Acid Dilution Buffer Investigator 24plex QS Kit (100)* Investigator ESSplex SE QS Kit (100)* Investigator IDplex Plus Kit* Primer mix, Fast Reaction Mix 2.0, Control DNA, allelic ladder 24plex QS, DNA size standard 550 (BTO), and nuclease free water Primer Mix, Fast Reaction Mix including Taq DNA Polymerase, Control DNA, Allelic Ladder ESSplex SE QS, DNA size standard 550 (BTO) and Nuclease-Free Water Primer mix, Fast Reaction Mix including HotStarTaq Plus DNA Polymerase, Control DNA, allelic ladder IDplex Plus, DNA size standard 550 (BTO), and nuclease-free water * Larger kit sizes available; please inquire. 387016 387116 382415 381575 381625 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 71 Product Investigator HDplex Kit (100) Investigator Triplex AFS QS Kit (400) Investigator Triplex DSF Kit (400) Investigator Argus X 12 Kit (25)* Investigator Argus Y 12 QS Kit (100) Investigator DIPplex Kit (100) Contents Primer mix, reaction mix, DNA Polymerase, Control DNA, allelic ladder, DNA size standard, and nuclease-free water Primer mix, reaction mix, DNA Polymerase, Control DNA, allelic ladder, DNA size standard, and nuclease-free water Primer mix, reaction mix, DNA Polymerase, Control DNA, allelic ladder, DNA size standard, and nuclease-free water Primer mix, reaction mix, DNA Polymerase, Control DNA, allelic ladder, DNA size standard, and nuclease-free water Primer mix, reaction mix, DNA Polymerase, Control DNA, allelic ladder, DNA size standard, and nuclease-free water Primer mix, reaction mix, DNA Polymerase, Control DNA, allelic ladder, DNA size standard, and nuclease-free water DNA extraction and purification QIAamp® DNA Investigator Kit (50) MinElute Reaction Cleanup Kit (50)* * Larger kit sizes available; please inquire. 50 QIAamp MinElute® Columns, Proteinase K, Carrier RNA, Buffers, Collection Tubes (2 ml) 50 MinElute Spin Columns, Buffers, Collection Tubes (2 ml) Cat. no. 381215 380317 380327 383213 383615 384015 56504 28004 72 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Product Contents Rotor-Gene Q consumables Strip Tubes and Caps, 0.1 ml (250) 250 strips of 4 tubes and caps for 1000 reactions Strip Tubes and Caps, 0.1 ml (2500) 10 x 250 strips of 4 tubes and caps for 10,000 reactions Rotor-Disc 72 (24) 24 individually wrapped discs for 1728 reactions Rotor-Disc 72 (240) 10 x 24 individually wrapped discs for 17,280 reactions Rotor-Disc 100 (30) 30 individually wrapped discs for 3000 reactions Rotor-Disc 100 (300) 10 x 30 individually wrapped discs for 30,000 reactions Rotor-Disc Heat Sealing Film (60) 60 films for sealing Rotor-Disc 100 or Rotor-Disc 72 discs Rotor-Disc Heat Sealing Film (600) 10 x 60 films for sealing Rotor-Disc 100 or Rotor-Disc 72 discs Rotor-Disc Heat Sealer 110 V Heat sealing instrument for use with Rotor-Discs; requires Rotor-Disc 72 or 100 Loading Block Rotor-Disc Heat Sealer 230 V Heat sealing instrument for use with Rotor-Discs; requires Rotor-Disc 72 or 100 Loading Block Cat. no. 981103 981106 981301 981303 981311 981313 981601 981604 9018898 9019725 For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor. Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 73 Limited License Agreement for Investigator Quantiplex Pro RGQ Kit Use of this product signifies the agreement of any purchaser or user of the product to the following terms: 1. The product may be used solely in accordance with the protocols provided with the product and this 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 protocols provided with the product, this handbook, and additional protocols available at www.qiagen.com. Some of these additional protocols have been provided by QIAGEN users for QIAGEN users. These protocols have not been thoroughly tested or optimized by QIAGEN. QIAGEN neither guarantees them nor warrants that they do not infringe the rights of third-parties. 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 www.qiagen.com. Trademarks: QIAGEN®, Sample to Insight®, QIAamp®, QIAgility®, 4NS1C®, Investigator®, MinElute®, Q-Bond®, QuantiNova®, Quantiplex®, QuantiTect®, Rotor-Disc®, Rotor-Gene® (QIAGEN Group); Applied Biosystems® (Life Technologies Corporation); TaqMan® (Roche Group). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law. HB-2521--001 © 2018 QIAGEN, all rights reserved. 74 Investigator Quantiplex Pro RGQ Kit Handbook 02/2018 Ordering www.qiagen.com/shop | Technical Support support.qiagen.com | Website www.qiagen.com I1n1ve1s2ti5g4a1tor0Q2u/a2n0ti1p8lex Pro RGQ Kit Handbook 02/2018 75Douglas Mcgarvey Adobe PDF Library 15.0