Setup and analysis Mic qpcr Cycler (magnetic induction cycler; Bio Molecular Systems)
General remark Runs on Mic (Bio Molecular Systems) can be set up and analysed either manually (described in part 1 of the manual) or using pre-defined assays (part 2). Troubleshooting for problems that might occur when using FTD assays on Mic is described in the third part of this manual. All descriptions in this setup manual are based on micpcr software version v1.12.0. 2
1. Manual setup and analysis 3
How to set up a run on Mic qpcr Cycler 1. Switch on the cycler first, then start the micpcr software (v1.12.0) 2. Click New and select Run to set all parameters manually, or Run from Template, if the FTD run profile is already saved on the computer. 4
How to set up a run on Mic qpcr Cycler 3. In the sidebar on the left, select Run Setup Run Profile. 4. Edit the cycling profiles to match the cycling steps listed in the manual of FTD kit. 5
How to set up a run on Mic qpcr Cycler 5. Make sure acquisition is switched on for all channels in the assay by clicking on the channel name. A camera icon indicates data acquisition in the respective channel. Data acquisition needs to be set to the end of the elongation step (indicated by a camera symbol). 6. Adjust gain settings can be left in default settings (AutoGain, all tubes). 6
How to set up a run on Mic qpcr Cycler 7. Make sure Temperature Control is set to Standard TAQ (v3) (default setting). 8. Adjust the reaction volume: 15 µl for FTlyo products, 25 µl for FTD liquid product line. Optional: To save these settings as a template, select Save as Template 7
How to set up a run on Mic qpcr Cycler 9. Insert the tubes into the rotor and make sure the tube clamp is in place. Load tubes filled with water in unused wells. 10. Close the lid, select the instrument in the software and Start Run. 8
How to analyse a run on the Mic qpcr Cycler (without assay function) Qualitative analysis 1. Assigning samples: The sample list can be edited before, during or after the run. Click on Run Setup Samples in the left side bar to open the sample editor. Enter sample names, assign the sample type and, if desired, change the display colour for the amplification plot. 2. Add a new analysis by clicking on the + icon next to Analysis Cycling and select Non-Assay and the channel you want to analyse. The analysis item will appear under Cycling in the sidebar. Note: Separate analyses should be created for each PPMix in the run. You can create multiple analyses of the same channel by repeating the steps described here. 9
How to analyse a run on Mic qpcr Cycler (without assay function) 3. In the panel Sample: Select all samples to be included in the analysis. De-selected samples will be greyed out. To hide samples but still have them included for analysis and the calculation of e.g., the automatic threshold, click on the eye icon next to the sample name. 10
How to analyse a run on Mic qpcr Cycler (without assay function) 4. In the panel Parameters, change the Method to Dynamic. If background fluorescence occurs during the first cycles, increase Ignore Cycles Before. Other parameters can be used as default. Note: If you do not want to use the Auto Set Threshold function, deselect this function by unchecking the tick box, and adjust the threshold by moving the red line in the panel Cycling Analysis. 5. When all assays are analysed, export the results either by creating a Report (click on + next to report in the left sidebar) or export the data as Excel workbook (Save as Excel workbook) 11
How to analyse a run on Mic qpcr Cycler (without assay function) Quantitative analysis 1. Assigning samples and standard concentrations: Click on Run Setup Samples in the left side bar to open the sample editor. a. Enter sample names and assign the sample type. b. Select the sample type Standard for all wells containing quantification standards (QS). c. Click on the icon Toggle between singleplex and multiplex standard to enable the standard function for all channels in one well d. Specify the concentration of each QS for all channels as listed in the manual of the respective FTD kit. 12
How to analyse a run on Mic qpcr Cycler (without assay function) 2. Add a new analysis by clicking on the + icon next to Absolute Quantification and select Non-Assay [Channel] 3. Click on the analysis name in the side bar and a small triangle/arrow will appear to the left of the assay. Click on this icon to expand the Cycling menu. 4. Select all samples and QS to be analysed in this analysis in the sample editor. Note: When running several FTD PPMixes during one run, create a separate analysis for each PPMix! 5. In the panel Parameters, change the Method to Dynamic. If background fluorescence occurs during the first cycles, increase Ignore Cycles Before. Other parameters can be used as default. Note: If you do not want to use the Auto Set Threshold function, deselect this function by unchecking the tick box, and adjust the threshold by moving the red line in the panel Cycling Analysis. 13
How to analyse a run on Mic qpcr Cycler (without assay function) 6. Click on the assay name again to view the results of the quantification. This view contains a plot of the standard curve and sample concentrations, the standard curve characteristics and results, and the results for the selected samples. Review if slope, efficiency and correlation coefficient match the target criteria given in the manual of the respective FTD kit. 14
2. Setup and analysis using the Assay function 15
Creating an assay Assay Setup 1. Start the micpcr software, click on the New icon and select Assay. 2. In the left sidebar, select Assay Setup - Information. 3. In the drop down menu Chemistry Type, select Hydrolysis Probes. 4. Enter the Name and Flurophore (or channel) for the first target of the assay in the field 5 Modifier. The other fields are not required for setting up the run. The tick box Contains Alleles should be switched off. 5. Additional targets can be added by clicking on the + icon next to Targets. 16
Creating an assay Assay Setup 6. To edit the run profile, select Assay Setup Profile. 7. Edit the cycling profiles to match the cycling steps listed in the manual of the FTD kit. 8. Make sure acquisition is switched on for all channels in the assay by clicking on the channel name. A camera icon indicates data acquisition in the respective channel. Data acquisition needs to be set to for the end of the elongation step (indicated by a camera symbol). 9. Adjust gain settings can be left in default settings (AutoGain, all tubes). 10. Make sure Temperature Control is set to Standard TAQ (v3) (default setting). 11. Adjust the reaction volume: 15 µl for FTlyo products, 25 µl for FTD liquid product line. 17
Creating an assay Analysis setup 12. Click on the the + icon next to the target and select Cycling. 13. Default criteria for assays with hydrolysis probes should appear as below: Note: It can be useful to increase Ignore Cycles Before if background fluorescence is high in the first cycles of the run (described in 3. Troubleshooting). 3. Save the assay. To be accessible from the assay list in the software, assay files need to be saved locally in the folder Assays of the software (default: [User]/My Documents/Bio Molecular Systems/micPCR/Assays) 18
How to setup a run on Mic qpcr Cycler using Assays 14. Switch on the cycler first, then start the micpcr software (v1.12.0) 15. Click New to create a new run. Add a saved assay by clicking on the + icon next to Assays in the left sidebar and select the assay(s) to be added to the run. If assay settings are not compatible because of different run profiles, a warning message will appear. 16. Insert the tubes into the rotor and make sure the tube clamp is in place. Load tubes filled with water into unused wells. Close the lid, select the instrument in the software and start the run. 19
How to analyse data using the assay function Qualitative analysis: 1. Select Run Setup Samples to open the sample editor and assign sample names and types. In the field Assay: Select the assay to be used from the drop down menu. 2. Add an analysis by clicking on the + icon next to Cycling and select the target you want to analyse. An analysis will be opened that contains only the samples to which the assay is assigned in the sample editor, analysed with the settings saved with the assay. If necessary analysis parameters can be modified as described in the first part of this manual. 3. When all assays are analysed, export the results either by creating a pdf Report (click on + next to report in the left sidebar) or export the data as Excel workbook (Save as Excel workbook) 20
Data analysis using the assay function Quantitative analysis: 1. Select Run Setup Samples to open the sample editor and assign sample names and types. Change the sample type Standard to all quantification standards. Click on the icon Toggle between singleplex and multiplex standards and enter the copy numbers for each channel as given in the manual of the corresponding FTD kit. 2. In the field assay: Select the assay to be used from the drop down menu. 21
Data analysis using the assay function 3. Add an analysis by clicking on the + icon next to Absolute Quantification and select the target you want to analyse. An analysis will be opened including only those samples to which the assay is assigned in the sample editor, analysed with the settings saved with the assay. Additionally, the results of the quantification standards are shown. If necessary analysis parameters can be modified as described in the first part of this manual. 4. When all assays are analysed, export the results either by creating a Report (click on + next to report in the left sidebar) or export the data as Excel workbook (Save as Excel workbook) 22
Troubleshooting 23
Troubleshooting In some cases, a combination of high background fluorescence during the first cycles of the run, and low signal intensity can require a modification of the default analysis parameters. Such problems can best be identified in the raw fluorescence curves under Data Cycling [Channel]. Samples with Mix C: high initial fluorescence, low signal intensity Adjust analysis parameters Mixes A+B: low background fluorescence and high signal to noise ratio Default analysis parameters work well Parameters to adjust in such a case (can be found in the Parameters panel of the Analysis): 1. Increase of number of cycles to ignore 2. Exclusion criteria a. Decrease Fluorescence cutoff level or b. Switch off entireley: select Exclusion None 24
Sloping or uneven baseline If the baseline is not flat or you observe a slope in the baseline, this can be caused by the wrong settings in the analysis parameters. Analysis Method LinRegPCR For all FTD assays, the analysis method must be set to Dynamic! Analysis Method Dynamic 25
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