iplex HS Melanoma Panel

Transcription

1 iplex HS Melanoma Panel This quick reference guide gives the assay protocol for the iplex HS Melanoma Panel. For complete information on equipment and reagents required, laboratory work areas and guidelines, preparing DNA samples, setting up the experiment in the software, and running reports, see the iplex HS Panels User Guide. For detailed instructions on desalting and dispensing analyte and acquiring data, see the applicable instrument user guide. All user guides are available on AgenaCx.com. A Note on Plate Types Users wishing to run samples in a 384-well format will perform PCR and SAP amplification in a 96-well plate, then transfer analyte to a 384-well plate before the iplex HS extension reaction. Table 1. Lab Area Activities Lab Area Activities 1 Isolation and dilution of DNA and preparation of the DNA source plate. Pre-PCR preparation, including preparation of the DNA reaction plate, preparation of the PCR 2 cocktail, and addition of the PCR cocktail to the reaction plate. Preparation of the SAP and extension cocktails. Thermocycling the reaction plate after addition of PCR cocktail; addition of the SAP cocktail to the 3 reaction plate and thermocycling; addition of the extension reaction cocktail to the reaction plate and thermocycling; desalting; nanodispensing; and data acquisition. 1. Preparing Sample DNA Plate The iplex HS Melanoma Panel requires at least 20 ng of DNA per sample (10 ng per PCR well). This is a 2-well PCR reaction panel; PCR-01 is then used for 7 extension wells and PCR-02 for 1 extension well. The recommended final volume of DNA input for each PCR reaction is 4 μl at 2.5ng/uL. IMPORTANT It is strongly recommended that the quality of the samples be checked using the Agena Bioscience Sample ID Panel. This is to ensure that amplifiable copies are used in the reaction, for optimal reaction conditions. Smaller or larger volumes of sample can be used by modifying the amount of water added. 1. To set up PCR-01 reaction plate, aliquot 4 μl of each sample into a specific well of a new 96-well plate. See Appendix for an example plate layout (Figure 1). 2. To set up PCR-02 reaction plate, aliquot 4 μl of each sample into a specific well of a new 96-well plate (Appendix Figure 2). Alternatively, these samples can be aliquoted into the same plate as PCR-01 plate, but in a separate row (e.g., Row B).

2 3. Visually inspect the individual wells from the bottom of the plate to confirm uniform and adequate DNA sample is present in every well before continuing. 4. Seal the plate and centrifuge at 2,000 x g for one minute. STOPPING POINT Purified genomic DNA can be stored at 4 C for up to 24 hours. To store DNA longer than 24 hours, we recommend storage at -20 C. Avoid repeated freezing and thawing of DNA. 2. PCR Reaction You will amplify DNA from your samples using the supplied PCR primers in a final reaction volume of 40 μl for PCR-01 and 10 μl for PCR-02. IMPORTANT Prepare the PCR cocktails and add to the reaction plate in Lab Area 2. Thermocycle the PCR reaction plate in Lab Area 3. Make sure all reagents are thawed completely at room temperature and enzymes are kept on ice. Make sure reagents are homogenized before taking aliquots. If plates were stored frozen prior to this step, make sure they are thawed completely at room temperature and spun down. 1) Prepare the two PCR cocktails in 1.5 ml tubes as shown in Table 2 and Table 3. Prepare more cocktail than the number of PCR reactions to be performed. Either prepare for one extra reaction or use a percentage extra to ensure sufficient overage is present to overcome typical pipetting variation. Table 2: PCR Cocktail for PCR-01 Reagent Per Well (μl) Final Concentration HPLC-grade water n/a 10X PCR Buffer 4.0 1X MgCl mm dntp Mix μm PCR Primer-P μm PCR Enzyme U/μL Final Volume PCR-01 Cocktail

3 Table 3: PCR Cocktail for PCR-02 Reagent Per Well (μl) Final Concentration HPLC-grade water 1.68 n/a 10X PCR Buffer 1.0 1X MgCl mm dntp Mix μm PCR Primer-P μm PCR Enzyme U/μL Final Volume PCR-02 Cocktail 6.0 2) Vortex the tube for 3 seconds and briefly centrifuge tube. 3) Transfer 36 μl of the PCR-01 cocktail into each sample well of the PCR-01 sample plate, for a final PCR reaction volume of 40 μl per well. 4) Transfer 6 μl of the PCR-02 cocktail into each sample well of the PCR-02 sample plate, for a final PCR reaction volume of 10 μl per well. 5) Seal the PCR reaction plates, vortex for 5 seconds at high speed, then centrifuge at 3200 x g for 5 seconds. 6) Visually inspect the individual wells from the bottom of the PCR reaction plates to confirm uniform and adequate cocktail solution is present in every well before continuing. 7) Thermocycle the PCR reaction plates using the following conditions: Initial Denaturation 95 C 2 minutes 1 cycle Cycled Template Denaturation 95 C 30 seconds Cycled Primer Annealing 56 C 30 seconds 45 cycles Cycled Primer Extension 72 C 1 minute Final Extension 72 C 5 minutes 1 cycle Hold 10 C Hold STOPPING POINT If not proceeding directly to the next step, the reaction plate should be sealed, and stored at 4 C (if storing for less than 24 hours), or at -20 C (if storing for more than 24 hours). Do not store for more than 2 weeks. 3

4 3. SAP Reaction IMPORTANT Prepare the SAP cocktail in Lab Area 2. Add the SAP cocktail to the reaction plates and thermocycle the plates in Lab Area 3. Make sure all reagents are thawed completely at room temperature and enzymes are kept on ice. Make sure all reagents are homogenized before taking aliquots. If plates were stored frozen prior to this step, may sure they are thawed completely at room temperature and spun down. 1) Centrifuge the reaction plates at 3200 x g for 5 seconds. 2) Prepare the SAP cocktail in a 1.5 ml tube as shown in Table 4 and Table 5. Prepare more cocktail than the number of SAP reactions to be performed. Either prepare for one extra reaction or use a percentage extra to ensure sufficient overage is present to overcome typical pipetting variation. Table 4: SAP Cocktail for 1 Sample (PCR-01 + PCR-02) Reagent Per Well Final Concentration (μl) HPLC-grade water 15.3 n/a 10X SAP Buffer X SAP Enzyme U/μL Final Volume SAP-01 Cocktail ) Vortex the tube for 3 seconds and briefly centrifuge. 4) Dispense 16 μl of SAP cocktail into each sample well of the PCR-01 reaction plate. 5) Dispense 4 μl of SAP cocktail into each sample well of the PCR-02 reaction plate. 6) Seal the reaction plates, vortex for 5 seconds at high speed, then centrifuge at 3200 x g for 5 seconds. 7) Visually inspect the individual wells from the bottom of the reaction plates to confirm uniform and adequate solution is present in every well before continuing. 8) Thermocycle the reaction plates using the following conditions: 37 C 40 minutes 85 C 5 minutes 10 C Hold 4

5 9) Dispense 7 μl of the PCR/SAP product into wells of a new microtiter plate, either 96-well or 384-well. See Appendix for an example 96-well plate layout (Figure 3). PCR/SAP-01 product will be dispensed into 7 extension wells PCR/SAP-02 product will be dispensed into 1 extension well. STOPPING POINT If not proceeding directly to the next step, the reaction plate should be sealed, and stored at 4 C (if storing for less than 24 hours), or at -20 C (if storing for more than 24 hours). Do not store for more than 2 weeks. 4. iplex HS Extension Reaction IMPORTANT Prepare the iplex HS extension reaction cocktails in Lab Area 2. Add the extension reaction cocktails to the reaction plate and thermocycle the plate in Lab Area 3. Make sure all reagents are thawed completely at room temperature and enzymes are kept on ice. Make sure all reagents are homogenized before taking aliquots. If plates were stored frozen prior to this step, make sure they are thawed completely at room temperature and spun down. 1) Centrifuge the reaction plate at 3200 x g for 5 seconds. 2) Prepare the iplex HS extension master mix in a 1.5 ml tube, as shown in Table 6. Prepare more master mix than the number of extension reactions to be performed. Either prepare for extra reactions or use a percentage extra to ensure sufficient overage is present to overcome typical pipetting variation. 5

6 Table 5. iplex HS Extension Master Mix Reagent Per Well (μl) Final concentration HPLC-grade water 0.62 n/a iplex Buffer Plus X iplex Pro Enzyme X Extension Master Mix Final Volume ) Vortex the tube for 3 seconds and briefly centrifuge. 4) Label eight 500 μl tubes from 1 to 8. 5) Prepare the extension reaction cocktails by adding extension master mix, extension primer, and termination mix to the 8 tubes, as specified in Table 7. Prepare more cocktail than the number of extension reactions to be performed. Either prepare for extra reactions or use a percentage extra to ensure sufficient overage is present to overcome typical pipetting variation. Table 6. iplex HS Extension Reaction Cocktails Extension Extension Master Mix Primer Termination Mix Extension Reaction Cocktail Final Volume Per Well (μl) Amount (μl) Tube Number same EXT W1 W1 2 same EXT W2 W2 3 same EXT W3 W3 4 same EXT W4 W4 5 same EXT W5 W5 6 same EXT W6 W6 7 same EXT W7 W7 8 same EXT W8 W8 6) Vortex the tubes for 3 seconds and briefly centrifuge. 7) Dispense 2 μl of extension reaction cocktail into the appropriate wells of the reaction plate. See example layout in appendix (Figure 4). 6

7 8) Seal the plate with thermal sealing film, vortex for 5 seconds at high speed, then centrifuge at 3200 x g for 5 seconds. 9) Visually inspect the individual wells from the bottom of the reaction plates to confirm uniform and adequate solution is present in every well before continuing. 10) Thermocycle the reaction plate using the following conditions. 95 C 30 seconds 95 C 5 seconds 52 C 5 seconds 5 cycles 40 cycles 80 C 5 seconds 72 C 3 minutes 10 C Hold STOPPING POINT If not proceeding directly to the next step, the reaction plate should be sealed, and stored at 4 C (if storing for less than 24 hours), or at -20 C (if storing for more than 24 hours). Do not store for more than 2 weeks. 5. Analyte Desalting/Dispensing and Data Acquisition Follow the instructions in the appropriate instrument user guides to desalt and dispense analyte onto SpectroCHIP Arrays and to acquire data with the MassARRAY Analyzer, using the following parameters. Processing on MassARRAY Nanodispenser RS1000 and MassARRAY Analyzer Parameter Water Addition Resin Recommended Value 96 plate: 41 ul 384 plate: 16 ul 96 plate: 15 mg dimple plate 384 plate: 6 mg dimple plate Nanodispenser RS1000 Settings Auto Tuning 7

8 Target Volume Volume Check Lower Limit/Upper Limit for Volume Check Dispense Speed Calibrant Dispense Speed 12 nl 8-20 nl 100 mm/sec 100 mm/sec Chiplinker Settings Terminator Chemistry Process Method iplex Genotyping+Area Analyzer 4 Settings Parameter File Filter Saturated Shots Use Calibration Wells Auto-Teach Geometry Turn off HV iplex_hs.par (acquisition parameters: 20/9/5/5) 8

9 Processing on MassARRAY System with Chip prep module Parameter Water Addition Recommended Value 96 plate: 41 ul ChipLinker Settings Terminator Chemistry Process Method iplex Genotyping+Area CPM/Analyzer 4 Settings Use Autotune SpectroCHIP CPM-96: not selected SpectroCHIP 96: selected Start Dispense Condition SpectroCHIP CPM-96: 600 SpectroCHIP 96: 500 Resin Volume 13 Sample Volume SpectroCHIP CPM-96: 30 SpectroCHIP 96: 7 Chip Heat SpectroCHIP CPM-96: selected; 30 SpectroCHIP 96: not selected Chemistry Parameter File iplex SpectroCHIP CPM-96: iplex_hs_cpm.par (acquisition parameters: 30/15/10/10) SpectroCHIP 96: iplex_hs.par (acquisition parameters: 20/9/5/5) Turn Off HV After Analysis Analyze Calibrant Pads Filter Saturated Shots 9

10 Appendix: Example Plate Layouts Figure 1. Example Sample Plate Layout for PCR-01 (numbers correspond to samples) Figure 2. Example Sample Plate Layout for PCR-02 (numbers correspond to samples) Figure 3. Example New PCR/SAP Plate: 7 μl PCR/SAP product per well (numbers correspond to samples) Dispense 7 ul PCR- 01/SAP product to Row A-G Dispense 7 ul PCR-02/SAP product to Row H 10

11 Figure 4. Example Extension Reaction Plate: 7 μl PCR/SAP product + 2 μl extension cocktail per well (numbers/colors correspond to a specific extension reaction cocktail) Patents and Trademarks MassARRAY and iplex are registered trademark of Agena Bioscience, Inc. Agena Bioscience is a trademark of Agena Bioscience, Inc. All other trademarks or service marks set forth herein are the property of their respective owners. Agena Bioscience s patented nucleic acid analysis by mass spectrometry methods and products are protected under United States patent rights including but not limited to; 6,440,705; 6,558,623; 6,730,517; 6,979,425; 6,994,969; 7,019,288; 7,025,933; 7,332,275; 7,390,672; 7,501,251; 7,888,127; 7,917,301; 8,003,317; 8,315,805; 8,349,566; 9,249,456; and 9,310,378, and patents pending including but not limited to US , and foreign counterparts including but not limited to EP B1, EP B1, EP B1, EP B1, EP B1, EP B1, and EP B1. [0217] The MassARRAY System and iplex HS Melanoma Panel are For Research Use Only. Not for use in diagnostic procedure. 5/29/18 11